Modified nucleotides and uses thereof

ABSTRACT

Disclosed herein, inter alia, are compounds, modified nucleotides, compositions, and methods of using the same.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.63/014,949, filed Apr. 24, 2020, and U.S. Provisional Application No.63/107,017, filed Oct. 29, 2020, which are incorporated herein byreference in their entirety and for all purposes.

BACKGROUND

DNA sequencing is a fundamental tool in biological and medical research;it is an essential technology for the paradigm of personalized precisionmedicine. Among various new DNA sequencing methods, sequencing bysynthesis (SBS) is the leading method for realizing the goal of the$1,000 genome. Accordingly, there is a need for modified nucleotides andnucleosides that are effectively recognized as substrates by DNApolymerases, that are efficiently and accurately incorporated intogrowing DNA chains during SBS. Disclosed herein, inter alia, aresolutions to these and other problems in the art.

BRIEF SUMMARY

In an aspect is provided a compound having the formula:

B¹ is a nucleobase. R⁷ is substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl. R⁸ issubstituted or unsubstituted alkyl. R¹ is independently hydrogen,halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂, —CHBr₂, —CHF₂, —CHI₂, —CH₂Cl,—CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H,—SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H,—NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃, —OCF₃, —OCBr₃, —OCI₃, —OCHCl₂,—OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl, —OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, a 5′-O-nucleoside protectinggroup, monophosphate moiety, polyphosphate moiety, or nucleic acidmoiety. R² is independently hydrogen, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃,—CHCl₂, —CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl; or apolymerase-compatible cleavable moiety.

In an aspect is provided a method for sequencing a nucleic acid,including (i) incorporating in series with a nucleic acid polymerase,within a reaction vessel, one of four different compounds into a primerto create an extension strand, wherein the primer is hybridized to thenucleic acid and wherein each of the four different compounds comprisesa unique detectable label; (ii) detecting the unique detectable label ofeach incorporated compound, so as to thereby identify each incorporatedcompound in the extension strand, thereby sequencing the nucleic acid;wherein each of the four different compounds is independently a compoundas described herein, including embodiments.

In an aspect is provided a method of incorporating a compound into aprimer, the method comprising combining a polymerase, a primerhybridized to nucleic acid template and the compound within a reactionvessel and allowing said polymerase to incorporate said compound intosaid primer thereby forming an extended primer, wherein said compound isa compound as described herein, including embodiments.

In an aspect is provided a nucleic acid polymerase complex including anucleic acid polymerase, wherein the nucleic acid polymerase is bound(e.g., non-covalently bound) to a compound described herein, includingembodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C. Depicted in FIG. 1A is the proposed fragmentationmechanism, wherein a base removes a hydrogen from the thiol (note, thedisulfide bond has already been reduced via a reducing agent). Theresulting transition state (TS) is then converted to a hydroxide and athioaldehyde. FIG. 1B shows the ylide and related resonance structuresfor different disulfide-containing reversible terminators. The greaternumber of resonant structures is a reflection of the greater electrondelocalization, which lowers the potential energy of the thioaldehyde,thereby stabilizing the molecule. A more stable thioaldehyde allows forfaster cleavage. FIG. 1C shows the chemical structure of a non-limitingexample of a synthesized modified nucleotide as described herein.

FIGS. 2A-2C. Cleavage kinetics of three nucleotides bearing reversibleterminators. The cleavage kinetics are improved relative to RT #1 and RT#2. FIG. 2B reports on the average cleavage halftime for RT #1, #2, and#3, where RT #1 is measured at an elevated temperature (65° C.), and RT#2 and RT #3 are measured at a lower temperature (55° C.) with identicalconcentrations of a cleaving agent, 1 mM THPP. FIG. 2C reports on theaverage cleavage halftime for RT #2, #3, #45, and #26 under identicalcleavage conditions, 0.1 mM THPP at 55° C.

FIG. 3 . Experimentally measured average cleavage halftimes underidentical conditions for RT #1, RT #2, and RT #3 as a function of theΔH. We found that in general, reducing the energetic burden on thesystem, i.e., reducing the enthalpy, correlates with faster cleavage.

DETAILED DESCRIPTION I. Definitions

The abbreviations used herein have their conventional meaning within thechemical and biological arts. The chemical structures and formulae setforth herein are constructed according to the standard rules of chemicalvalency known in the chemical arts.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents that would result from writing thestructure from right to left, e.g., —CH₂O— is equivalent to —OCH₂—.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight (i.e., unbranched) or branchedcarbon chain (or carbon), or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include mono-, di- andmultivalent radicals. The alkyl may include a designated number ofcarbons (e.g., C₁-C₁₀ means one to ten carbons). In embodiments, thealkyl is fully saturated. In embodiments, the alkyl is monounsaturated.In embodiments, the alkyl is polyunsaturated. Alkyl is an uncyclizedchain. Examples of saturated hydrocarbon radicals include, but are notlimited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl,t-butyl, isobutyl, sec-butyl, methyl, homologs and isomers of, forexample, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Anunsaturated alkyl group is one having one or more double bonds or triplebonds. Examples of unsaturated alkyl groups include, but are not limitedto, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,3-butynyl, and the higher homologs and isomers. An alkoxy is an alkylattached to the remainder of the molecule via an oxygen linker (—O—). Analkyl moiety may be an alkenyl moiety. An alkyl moiety may be an alkynylmoiety. An alkyl moiety may be fully saturated. An alkenyl may includemore than one double bond and/or one or more triple bonds in addition tothe one or more double bonds. An alkynyl may include more than onetriple bond and/or one or more double bonds in addition to the one ormore triple bonds. An alkenyl includes one or more double bonds. Analkynyl includes one or more triple bonds.

The term “alkylene,” by itself or as part of another substituent, means,unless otherwise stated, a divalent radical derived from an alkyl, asexemplified, but not limited by, —CH₂CH₂CH₂CH₂—. Typically, an alkyl (oralkylene) group will have from 1 to 24 carbon atoms, with those groupshaving 10 or fewer carbon atoms being preferred herein. A “lower alkyl”or “lower alkylene” is a shorter chain alkyl or alkylene group,generally having eight or fewer carbon atoms. The term “alkenylene,” byitself or as part of another substituent, means, unless otherwisestated, a divalent radical derived from an alkene. The term “alkynylene”by itself or as part of another substituent, means, unless otherwisestated, a divalent radical derived from an alkyne. The term “alkynylene”by itself or as part of another substituent, means, unless otherwisestated, a divalent radical derived from an alkyne. In embodiments, thealkylene is fully saturated. In embodiments, the alkylene ismonounsaturated. In embodiments, the alkylene is polyunsaturated. Analkenylene includes one or more double bonds. An alkynylene includes oneor more triple bonds.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcombinations thereof, including at least one carbon atom and at leastone heteroatom (e.g., O, N, P, Si, and S), and wherein the nitrogen andsulfur atoms may optionally be oxidized, and the nitrogen heteroatom mayoptionally be quaternized. The heteroatom(s) (e.g., O, N, S, Si, or P)may be placed at any interior position of the heteroalkyl group or atthe position at which the alkyl group is attached to the remainder ofthe molecule. Heteroalkyl is an uncyclized chain. Examples include, butare not limited to: —CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃,—CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—S—CH₂, —S(O)—CH₃,—CH₂—CH₂—S(O)₂—CH₃, —CH═CHO—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃,—CH═CH—N(CH₃)—CH₃, —O—CH₃, —O—CH₂—CH₃, and —CN. Up to two or threeheteroatoms may be consecutive, such as, for example, —CH₂—NH—OCH₃ and—CH₂—O—Si(CH₃)₃. A heteroalkyl moiety may include one heteroatom (e.g.,O, N, S, Si, or P). A heteroalkyl moiety may include two optionallydifferent heteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl moietymay include three optionally different heteroatoms (e.g., O, N, S, Si,or P). A heteroalkyl moiety may include four optionally differentheteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl moiety may includefive optionally different heteroatoms (e.g., O, N, S, Si, or P). Aheteroalkyl moiety may include up to 8 optionally different heteroatoms(e.g., O, N, S, Si, or P). The term “heteroalkenyl,” by itself or incombination with another term, means, unless otherwise stated, aheteroalkyl including at least one double bond. A heteroalkenyl mayoptionally include more than one double bond and/or one or more triplebonds in additional to the one or more double bonds. The term“heteroalkynyl,” by itself or in combination with another term, means,unless otherwise stated, a heteroalkyl including at least one triplebond. A heteroalkynyl may optionally include more than one triple bondand/or one or more double bonds in additional to the one or more triplebonds. In embodiments, the heteroalkyl is fully saturated. Inembodiments, the heteroalkyl is monounsaturated. In embodiments, theheteroalkyl is polyunsaturated.

Similarly, the term “heteroalkylene,” by itself or as part of anothersubstituent, means, unless otherwise stated, a divalent radical derivedfrom heteroalkyl, as exemplified, but not limited by,—CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylenegroups, heteroatoms can also occupy either or both of the chain termini(e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, andthe like). Still further, for alkylene and heteroalkylene linkinggroups, no orientation of the linking group is implied by the directionin which the formula of the linking group is written. For example, theformula —C(O)₂R′— represents both —C(O)₂R′— and —R′C(O)₂—. As describedabove, heteroalkyl groups, as used herein, include those groups that areattached to the remainder of the molecule through a heteroatom, such as—C(O)R′, —C(O)NR′, —NR′R″, —OR′, —SR′, and/or —SO₂R′. Where“heteroalkyl” is recited, followed by recitations of specificheteroalkyl groups, such as —NR′R″ or the like, it will be understoodthat the terms heteroalkyl and —NR′R″ are not redundant or mutuallyexclusive. Rather, the specific heteroalkyl groups are recited to addclarity. Thus, the term “heteroalkyl” should not be interpreted hereinas excluding specific heteroalkyl groups, such as —NR′R″ or the like.The term “heteroalkenylene,” by itself or as part of anothersubstituent, means, unless otherwise stated, a divalent radical derivedfrom a heteroalkene. The term “heteroalkynylene” by itself or as part ofanother substituent, means, unless otherwise stated, a divalent radicalderived from a heteroalkyne. In embodiments, the heteroalkylene is fullysaturated. In embodiments, the heteroalkylene is monounsaturated. Inembodiments, the heteroalkylene is polyunsaturated. A heteroalkenyleneincludes one or more double bonds. A heteroalkynylene includes one ormore triple bonds.

The terms “cycloalkyl” and “heterocycloalkyl,” by themselves or incombination with other terms, mean, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl,” respectively. Cycloalkyl andheterocycloalkyl are not aromatic. Additionally, for heterocycloalkyl, aheteroatom can occupy the position at which the heterocycle is attachedto the remainder of the molecule. Examples of cycloalkyl include, butare not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like. A “cycloalkylene” and a“heterocycloalkylene,” alone or as part of another substituent, means adivalent radical derived from a cycloalkyl and heterocycloalkyl,respectively. In embodiments, the cycloalkyl is fully saturated. Inembodiments, the cycloalkyl is monounsaturated. In embodiments, thecycloalkyl is polyunsaturated. In embodiments, the heterocycloalkyl isfully saturated. In embodiments, the heterocycloalkyl ismonounsaturated. In embodiments, the heterocycloalkyl ispolyunsaturated.

In embodiments, the term “cycloalkyl” means a monocyclic, bicyclic, or amulticyclic cycloalkyl ring system. In embodiments, monocyclic ringsystems are cyclic hydrocarbon groups containing from 3 to 8 carbonatoms, where such groups can be saturated or unsaturated, but notaromatic. In embodiments, cycloalkyl groups are fully saturated. Inembodiments, a bicyclic or multicyclic cycloalkyl ring system refers tomultiple rings fused together or multiple spirocyclic rings wherein atleast one of the fused or spirocyclic rings is a cycloalkyl ring andwherein the multiple rings are attached to the parent molecular moietythrough any carbon atom contained within a cycloalkyl ring of themultiple rings.

In embodiments, a cycloalkyl is a cycloalkenyl. The term “cycloalkenyl”is used in accordance with its plain ordinary meaning. In embodiments, acycloalkenyl is a monocyclic, bicyclic, or a multicyclic cycloalkenylring system. In embodiments, a bicyclic or multicyclic cycloalkenyl ringsystem refers to multiple rings fused together or multiple spirocyclicrings wherein at least one of the fused or spirocyclic rings is acycloalkenyl ring and wherein the multiple rings are attached to theparent molecular moiety through any carbon atom contained within acycloalkenyl ring of the multiple rings.

In embodiments, the term “heterocycloalkyl” means a monocyclic,bicyclic, or a multicyclic heterocycloalkyl ring system. In embodiments,heterocycloalkyl groups are fully saturated. In embodiments, a bicyclicor multicyclic heterocycloalkyl ring system refers to multiple ringsfused together or multiple spirocyclic rings wherein at least one of thefused or spirocyclic rings is a heterocycloalkyl ring and wherein themultiple rings are attached to the parent molecular moiety through anyatom contained within a heterocycloalkyl ring of the multiple rings.

In embodiments, the term “cycloalkyl” means a monocyclic, bicyclic, or amulticyclic cycloalkyl ring system. In embodiments, monocyclic ringsystems are cyclic hydrocarbon groups containing from 3 to 8 carbonatoms, where such groups can be saturated or unsaturated, but notaromatic. In embodiments, cycloalkyl groups are fully saturated.Examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl,cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, andcyclooctyl. Bicyclic cycloalkyl ring systems are bridged monocyclicrings or fused bicyclic rings. In embodiments, bridged monocyclic ringscontain a monocyclic cycloalkyl ring where two non adjacent carbon atomsof the monocyclic ring are linked by an alkylene bridge of between oneand three additional carbon atoms (i.e., a bridging group of the form(CH₂)_(w), where w is 1, 2, or 3). Representative examples of bicyclicring systems include, but are not limited to, bicyclo[3.1.1]heptane,bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, bicyclo[3.2.2]nonane,bicyclo[3.3.1]nonane, and bicyclo[4.2.1]nonane. In embodiments, fusedbicyclic cycloalkyl ring systems contain a monocyclic cycloalkyl ringfused to either a monocyclic cycloalkyl, a monocyclic cycloalkenyl, or amonocyclic heterocyclyl. In embodiments, the bridged or fused bicycliccycloalkyl is attached to the parent molecular moiety through any carbonatom contained within the monocyclic cycloalkyl ring. In embodiments,cycloalkyl groups are optionally substituted with one or two groupswhich are independently oxo or thia. In embodiments, the fused bicycliccycloalkyl is a 5 or 6 membered monocyclic cycloalkyl ring fused toeither a 5 or 6 membered monocyclic cycloalkyl, a 5 or 6 memberedmonocyclic cycloalkenyl, or a 5 or 6 membered monocyclic heterocyclyl,wherein the fused bicyclic cycloalkyl is optionally substituted by oneor two groups which are independently oxo or thia. In embodiments,multicyclic cycloalkyl ring systems are a monocyclic cycloalkyl ring(base ring) fused to either (i) one ring system selected from the groupconsisting of a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and abicyclic heterocyclyl; or (ii) two other ring systems independentlyselected from the group consisting of a monocyclic or bicycliccycloalkyl, a monocyclic or bicyclic cycloalkenyl, and a monocyclic orbicyclic heterocyclyl. In embodiments, the multicyclic cycloalkyl isattached to the parent molecular moiety through any carbon atomcontained within the base ring. In embodiments, multicyclic cycloalkylring systems are a monocyclic cycloalkyl ring (base ring) fused toeither (i) one ring system selected from the group consisting of abicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclicheterocyclyl; or (ii) two other ring systems independently selected fromthe group consisting of a monocyclic cycloalkyl, a monocycliccycloalkenyl, and a monocyclic heterocyclyl.

In embodiments, a cycloalkyl is a cycloalkenyl. The term “cycloalkenyl”is used in accordance with its plain ordinary meaning. In embodiments, acycloalkenyl is a monocyclic, bicyclic, or a multicyclic cycloalkenylring system. In embodiments, monocyclic cycloalkenyl ring systems arecyclic hydrocarbon groups containing from 3 to 8 carbon atoms, wheresuch groups are unsaturated (i.e., containing at least one annularcarbon carbon double bond), but not aromatic. Examples of monocycliccycloalkenyl ring systems include cyclopentenyl and cyclohexenyl. Inembodiments, bicyclic cycloalkenyl rings are bridged monocyclic rings ora fused bicyclic rings. In embodiments, bridged monocyclic rings containa monocyclic cycloalkenyl ring where two non adjacent carbon atoms ofthe monocyclic ring are linked by an alkylene bridge of between one andthree additional carbon atoms (i.e., a bridging group of the form(CH₂)_(w), where w is 1, 2, or 3). Representative examples of bicycliccycloalkenyls include, but are not limited to, norbornenyl andbicyclo[2.2.2]oct 2 enyl. In embodiments, fused bicyclic cycloalkenylring systems contain a monocyclic cycloalkenyl ring fused to either amonocyclic cycloalkyl, a monocyclic cycloalkenyl, or a monocyclicheterocyclyl. In embodiments, the bridged or fused bicyclic cycloalkenylis attached to the parent molecular moiety through any carbon atomcontained within the monocyclic cycloalkenyl ring. In embodiments,cycloalkenyl groups are optionally substituted with one or two groupswhich are independently oxo or thia. In embodiments, multicycliccycloalkenyl rings contain a monocyclic cycloalkenyl ring (base ring)fused to either (i) one ring system selected from the group consistingof a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclicheterocyclyl; or (ii) two ring systems independently selected from thegroup consisting of a monocyclic or bicyclic cycloalkyl, a monocyclic orbicyclic cycloalkenyl, and a monocyclic or bicyclic heterocyclyl. Inembodiments, the multicyclic cycloalkenyl is attached to the parentmolecular moiety through any carbon atom contained within the base ring.In embodiments, multicyclic cycloalkenyl rings contain a monocycliccycloalkenyl ring (base ring) fused to either (i) one ring systemselected from the group consisting of a bicyclic cycloalkyl, a bicycliccycloalkenyl, and a bicyclic heterocyclyl; or (ii) two ring systemsindependently selected from the group consisting of a monocycliccycloalkyl, a monocyclic cycloalkenyl, and a monocyclic heterocyclyl.

In embodiments, a heterocycloalkyl is a heterocyclyl. The term“heterocyclyl” as used herein, means a monocyclic, bicyclic, ormulticyclic heterocycle. The heterocyclyl monocyclic heterocycle is a 3,4, 5, 6 or 7 membered ring containing at least one heteroatomindependently selected from the group consisting of O, N, and S wherethe ring is saturated or unsaturated, but not aromatic. The 3 or 4membered ring contains one heteroatom selected from the group consistingof O, N and S. The 5 membered ring can contain zero or one double bondand one, two or three heteroatoms selected from the group consisting ofO, N and S. The 6 or 7 membered ring contains zero, one or two doublebonds and one, two or three heteroatoms selected from the groupconsisting of O, N and S. The heterocyclyl monocyclic heterocycle isconnected to the parent molecular moiety through any carbon atom or anynitrogen atom contained within the heterocyclyl monocyclic heterocycle.Representative examples of heterocyclyl monocyclic heterocycles include,but are not limited to, azetidinyl, azepanyl, aziridinyl, diazepanyl,1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl,imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl,isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl,oxadiazolidinyl, oxazolinyl, oxazolidinyl, piperazinyl, piperidinyl,pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl,tetrahydrofuranyl, tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl,thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxidothiomorpholinyl(thiomorpholine sulfone), thiopyranyl, and trithianyl. The heterocyclylbicyclic heterocycle is a monocyclic heterocycle fused to either amonocyclic cycloalkyl, a monocyclic cycloalkenyl, or a monocyclicheterocycle. The heterocyclyl bicyclic heterocycle is connected to theparent molecular moiety through any carbon atom or any nitrogen atomcontained within the monocyclic heterocycle portion of the bicyclic ringsystem. Representative examples of bicyclic heterocyclyls include, butare not limited to, 2,3-dihydrobenzofuran-2-yl,2,3-dihydrobenzofuran-3-yl, indolin-1-yl, indolin-2-yl, indolin-3-yl,2,3-dihydrobenzothien-2-yl, decahydroquinolinyl, decahydroisoquinolinyl,octahydro-1H-indolyl, and octahydrobenzofuranyl. In embodiments,heterocyclyl groups are optionally substituted with one or two groupswhich are independently oxo or thia. In certain embodiments, thebicyclic heterocyclyl is a 5 or 6 membered monocyclic heterocyclyl ringfused to a 5 or 6 membered monocyclic cycloalkyl, a 5 or 6 memberedmonocyclic cycloalkenyl, or a 5 or 6 membered monocyclic heterocyclyl,wherein the bicyclic heterocyclyl is optionally substituted by one ortwo groups which are independently oxo or thia. Multicyclic heterocyclylring systems are a monocyclic heterocyclyl ring (base ring) fused toeither (i) one ring system selected from the group consisting of abicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclicheterocyclyl; or (ii) two other ring systems independently selected fromthe group consisting of a monocyclic or bicyclic cycloalkyl, amonocyclic or bicyclic cycloalkenyl, and a monocyclic or bicyclicheterocyclyl. The multicyclic heterocyclyl is attached to the parentmolecular moiety through any carbon atom or nitrogen atom containedwithin the base ring. In embodiments, multicyclic heterocyclyl ringsystems are a monocyclic heterocyclyl ring (base ring) fused to either(i) one ring system selected from the group consisting of a bicycliccycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or(ii) two other ring systems independently selected from the groupconsisting of a monocyclic cycloalkyl, a monocyclic cycloalkenyl, and amonocyclic heterocyclyl.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C1-C4)alkyl” includes, but is not limited to, fluoromethyl,difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl,3-bromopropyl, and the like.

The term “acyl” means, unless otherwise stated, —C(O)R where R is asubstituted or unsubstituted alkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent, which can be a single ring ormultiple rings (preferably from 1 to 3 rings) that are fused together(i.e., a fused ring aryl) or linked covalently. A fused ring aryl refersto multiple rings fused together wherein at least one of the fused ringsis an aryl ring. In embodiments, a fused ring aryl refers to multiplerings fused together wherein at least one of the fused rings is an arylring and wherein the multiple rings are attached to the parent molecularmoiety through any carbon atom contained within an aryl ring of themultiple rings. The term “heteroaryl” refers to aryl groups (or rings)that contain at least one heteroatom such as N, O, or S, wherein thenitrogen and sulfur atoms are optionally oxidized, and the nitrogenatom(s) are optionally quaternized. Thus, the term “heteroaryl” includesfused ring heteroaryl groups (i.e., multiple rings fused togetherwherein at least one of the fused rings is a heteroaromatic ring). Inembodiments, the term “heteroaryl” includes fused ring heteroaryl groups(i.e., multiple rings fused together wherein at least one of the fusedrings is a heteroaromatic ring and wherein the multiple rings areattached to the parent molecular moiety through any atom containedwithin a heteroaromatic ring of the multiple rings). A 5,6-fused ringheteroarylene refers to two rings fused together, wherein one ring has 5members and the other ring has 6 members, and wherein at least one ringis a heteroaryl ring. Likewise, a 6,6-fused ring heteroarylene refers totwo rings fused together, wherein one ring has 6 members and the otherring has 6 members, and wherein at least one ring is a heteroaryl ring.And a 6,5-fused ring heteroarylene refers to two rings fused together,wherein one ring has 6 members and the other ring has 5 members, andwherein at least one ring is a heteroaryl ring. A heteroaryl group canbe attached to the remainder of the molecule through a carbon orheteroatom. Non-limiting examples of aryl and heteroaryl groups includephenyl, naphthyl, pyrrolyl, pyrazolyl, pyridazinyl, triazinyl,pyrimidinyl, imidazolyl, pyrazinyl, purinyl, oxazolyl, isoxazolyl,thiazolyl, furyl, thienyl, pyridyl, pyrimidyl, benzothiazolyl,benzoxazoyl benzimidazolyl, benzofuran, isobenzofuranyl, indolyl,isoindolyl, benzothiophenyl, isoquinolyl, quinoxalinyl, quinolyl,1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl,3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl,4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Substituents for each of the above notedaryl and heteroaryl ring systems are selected from the group ofacceptable substituents described below. An “arylene” and a“heteroarylene,” alone or as part of another substituent, mean adivalent radical derived from an aryl and heteroaryl, respectively. Aheteroaryl group substituent may be —O— bonded to a ring heteroatomnitrogen.

Spirocyclic rings are two or more rings wherein adjacent rings areattached through a single atom. The individual rings within spirocyclicrings may be identical or different. Individual rings in spirocyclicrings may be substituted or unsubstituted and may have differentsubstituents from other individual rings within a set of spirocyclicrings. Possible substituents for individual rings within spirocyclicrings are the possible substituents for the same ring when not part ofspirocyclic rings (e.g., substituents for cycloalkyl or heterocycloalkylrings). Spirocyclic rings may be substituted or unsubstitutedcycloalkyl, substituted or unsubstituted cycloalkylene, substituted orunsubstituted heterocycloalkyl or substituted or unsubstitutedheterocycloalkylene and individual rings within a spirocyclic ring groupmay be any of the immediately previous list, including having all ringsof one type (e.g., all rings being substituted heterocycloalkylenewherein each ring may be the same or different substitutedheterocycloalkylene). When referring to a spirocyclic ring system,heterocyclic spirocyclic rings means a spirocyclic rings wherein atleast one ring is a heterocyclic ring and wherein each ring may be adifferent ring. When referring to a spirocyclic ring system, substitutedspirocyclic rings means that at least one ring is substituted and eachsubstituent may optionally be different.

The symbol “

” denotes the point of attachment of a chemical moiety to the remainderof a molecule or chemical formula. The term “oxo,” as used herein, meansan oxygen that is double bonded to a carbon atom.

The term “alkylarylene” as an arylene moiety covalently bonded to analkylene moiety (also referred to herein as an alkylene linker). Inembodiments, the alkylarylene group has the formula:

An alkylarylene moiety may be substituted (e.g., with a substituentgroup) on the alkylene moiety or the arylene linker (e.g., at carbons 2,3, 4, or 6) with halogen, oxo, —N₃, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN, —CHO,—OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₂CH₃, —SO₃H, —OSO₃H, —SO₂NH₂,—NHNH₂, —ONH₂, —NHC(O)NHNH₂, substituted or unsubstituted C₁-C₅ alkyl orsubstituted or unsubstituted 2 to 5 membered heteroalkyl). Inembodiments, the alkylarylene is unsubstituted.

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “cycloalkyl,”“heterocycloalkyl,” “aryl,” and “heteroaryl”) includes both substitutedand unsubstituted forms of the indicated radical. Preferred substituentsfor each type of radical are provided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′, halogen,—SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —NR′NR″R′″,—ONR′R″, —NR′C(O)NR″NR′″R″″, —CN, —NO₂, —NR′SO₂R″, —NR′C(O)R″,—NR′C(O)—OR″, —NR′OR″, in a number ranging from zero to (2m′+1), wherem′ is the total number of carbon atoms in such radical. R, R′, R″, R′″,and R″″ each preferably independently refer to hydrogen, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl (e.g., aryl substituted with 1-3 halogens),substituted or unsubstituted heteroaryl, substituted or unsubstitutedalkyl, alkoxy, or thioalkoxy groups, or arylalkyl groups. When acompound described herein includes more than one R group, for example,each of the R groups is independently selected as are each R′, R″, R′″,and R″″ group when more than one of these groups is present. When R′ andR″ are attached to the same nitrogen atom, they can be combined with thenitrogen atom to form a 4-, 5-, 6-, or 7-membered ring. For example,—NR′R″ includes, but is not limited to, 1-pyrrolidinyl and4-morpholinyl. From the above discussion of substituents, one of skillin the art will understand that the term “alkyl” is meant to includegroups including carbon atoms bound to groups other than hydrogengroups, such as haloalkyl (e.g., —CF₃ and —CH₂CF₃) and acyl (e.g.,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are varied and areselected from, for example: —OR′, —NR′R″, —SR′, halogen, —SiR′R″R′″,—OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′,—NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR′″,—S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —NR′NR″R′″, —ONR′R″,—NR′C(O)NR″NR′″R″″, —CN, —NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy,and fluoro(C₁-C₄)alkyl, —NR′SO₂R″, —NR′C(O)R″, —NR′C(O)—OR″, —NR′OR″, ina number ranging from zero to the total number of open valences on thearomatic ring system; and where R′, R″, R′″, and R″″ are preferablyindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl. When a compound described herein includes more than one Rgroup, for example, each of the R groups is independently selected asare each R′, R″, R′″, and R″″ groups when more than one of these groupsis present.

As used herein, the term “associated” or “associated with” can mean thattwo or more species are identifiable as being co-located at a point intime. An association can mean that two or more species are or werewithin a similar container. An association can be an informaticsassociation, where for example digital information regarding two or morespecies is stored and can be used to determine that one or more of thespecies were co-located at a point in time. An association can also be aphysical association.

Substituents for rings (e.g., cycloalkyl, heterocycloalkyl, aryl,heteroaryl, cycloalkylene, heterocycloalkylene, arylene, orheteroarylene) may be depicted as substituents on the ring rather thanon a specific atom of a ring (commonly referred to as a floatingsubstituent). In such a case, the substituent may be attached to any ofthe ring atoms (obeying the rules of chemical valency) and in the caseof fused rings or spirocyclic rings, a substituent depicted asassociated with one member of the fused rings or spirocyclic rings (afloating substituent on a single ring), may be a substituent on any ofthe fused rings or spirocyclic rings (a floating substituent on multiplerings). When a substituent is attached to a ring, but not a specificatom (a floating substituent), and a subscript for the substituent is aninteger greater than one, the multiple substituents may be on the sameatom, same ring, different atoms, different fused rings, differentspirocyclic rings, and each substituent may optionally be different.Where a point of attachment of a ring to the remainder of a molecule isnot limited to a single atom (a floating substituent), the attachmentpoint may be any atom of the ring and in the case of a fused ring orspirocyclic ring, any atom of any of the fused rings or spirocyclicrings while obeying the rules of chemical valency. Where a ring, fusedrings, or spirocyclic rings contain one or more ring heteroatoms and thering, fused rings, or spirocyclic rings are shown with one more floatingsubstituents (including, but not limited to, points of attachment to theremainder of the molecule), the floating substituents may be bonded tothe heteroatoms. Where the ring heteroatoms are shown bound to one ormore hydrogens (e.g., a ring nitrogen with two bonds to ring atoms and athird bond to a hydrogen) in the structure or formula with the floatingsubstituent, when the heteroatom is bonded to the floating substituent,the substituent will be understood to replace the hydrogen, whileobeying the rules of chemical valency.

Two or more substituents may optionally be joined to form aryl,heteroaryl, cycloalkyl, or heterocycloalkyl groups. Such so-calledring-forming substituents are typically, though not necessarily, foundattached to a cyclic base structure. In one embodiment, the ring-formingsubstituents are attached to adjacent members of the base structure. Forexample, two ring-forming substituents attached to adjacent members of acyclic base structure create a fused ring structure. In anotherembodiment, the ring-forming substituents are attached to a singlemember of the base structure. For example, two ring-forming substituentsattached to a single member of a cyclic base structure create aspirocyclic structure. In yet another embodiment, the ring-formingsubstituents are attached to non-adjacent members of the base structure.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally form a ring of the formula -T-C(O)—(CRR′)_(q)—U—, whereinT and U are independently —NR—, —O—, —CRR′—, or a single bond, and q isan integer from 0 to 3. Alternatively, two of the substituents onadjacent atoms of the aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula -A-(CH₂)_(r)—B—, wherein A and B areindependently —CRR′—, —O—, —NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′—, or asingle bond, and r is an integer from 1 to 4. One of the single bonds ofthe new ring so formed may optionally be replaced with a double bond.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula —(CRR′)_(s)—X′— (C″R″R′″)_(d)—, where s and d are independentlyintegers from 0 to 3, and X′ is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or—S(O)₂NR′—. The substituents R, R′, R″, and R′″ are preferablyindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl.

As used herein, the terms “heteroatom” or “ring heteroatom” are meant toinclude oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), andsilicon (Si).

A “substituent group,” as used herein, means a group selected from thefollowing moieties:

-   -   (A) oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂, —CHBr₂,        —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂,        —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,        —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,        —OCCl₃, —OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂,        —OCH₂Cl, —OCH₂Br, —OCH₂I, —OCH₂F, —N₃, unsubstituted alkyl        (e.g., C₁-C₈ alkyl, C₁-C₆ alkyl, or C₁-C₄ alkyl), unsubstituted        heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered        heteroalkyl, or 2 to 4 membered heteroalkyl), unsubstituted        cycloalkyl (e.g., C₃-C₈ cycloalkyl, C₃-C₆ cycloalkyl, or C₅-C₆        cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3 to 8        membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or        5 to 6 membered heterocycloalkyl), unsubstituted aryl (e.g.,        C₆-C₁₀ aryl, C₁₀ aryl, or phenyl), or unsubstituted heteroaryl        (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl,        or 5 to 6 membered heteroaryl), and    -   (B) alkyl (e.g., C₁-C₈ alkyl, C₁-C₆ alkyl, or C₁-C₄ alkyl),        heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered        heteroalkyl, or 2 to 4 membered heteroalkyl), cycloalkyl (e.g.,        C₃-C₈ cycloalkyl, C₃-C₆ cycloalkyl, or C₅-C₆ cycloalkyl),        heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6        membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl),        aryl (e.g., C₆-C₁₀ aryl, C₁₀ aryl, or phenyl), heteroaryl (e.g.,        5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to        6 membered heteroaryl), substituted with at least one        substituent selected from:        -   (i) oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂, —CHBr₂,            —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂,            —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,            —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H,            —NHC(O)OH, —NHOH, —OCCl₃, —OCF₃, —OCBr₃, —OCI₃, —OCHCl₂,            —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl, —OCH₂Br, —OCH₂I, —OCH₂F,            —N₃, unsubstituted alkyl (e.g., C₁-C₈ alkyl, C₁-C₆ alkyl, or            C₁-C₄ alkyl), unsubstituted heteroalkyl (e.g., 2 to 8            membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4            membered heteroalkyl), unsubstituted cycloalkyl (e.g., C₃-C₈            cycloalkyl, C₃-C₆ cycloalkyl, or C₅-C₆ cycloalkyl),            unsubstituted heterocycloalkyl (e.g., 3 to 8 membered            heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to            6 membered heterocycloalkyl), unsubstituted aryl (e.g.,            C₆-C₁₀ aryl, C₁₀ aryl, or phenyl), or unsubstituted            heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9            membered heteroaryl, or 5 to 6 membered heteroaryl), and        -   (ii) alkyl (e.g., C₁-C₈ alkyl, C₁-C₆ alkyl, or C₁-C₄ alkyl),            heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6            membered heteroalkyl, or 2 to 4 membered heteroalkyl),            cycloalkyl (e.g., C₃-C₈ cycloalkyl, C₃-C₆ cycloalkyl, or            C₅-C₆ cycloalkyl), heterocycloalkyl (e.g., 3 to 8 membered            heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to            6 membered heterocycloalkyl), aryl (e.g., C₆-C₁₀ aryl, C₁₀            aryl, or phenyl), heteroaryl (e.g., 5 to 10 membered            heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered            heteroaryl), substituted with at least one substituent            selected from:            -   (a) oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂,                —CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN,                —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H,                —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂,                —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃, —OCF₃,                —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂,                —OCH₂Cl, —OCH₂Br, —OCH₂I, —OCH₂F, —N₃, unsubstituted                alkyl (e.g., C₁-C₈ alkyl, C₁-C₆ alkyl, or C₁-C₄ alkyl),                unsubstituted heteroalkyl (e.g., 2 to 8 membered                heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4                membered heteroalkyl), unsubstituted cycloalkyl (e.g.,                C₃-C₈ cycloalkyl, C₃-C₆ cycloalkyl, or C₅-C₆                cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3 to                8 membered heterocycloalkyl, 3 to 6 membered                heterocycloalkyl, or 5 to 6 membered heterocycloalkyl),                unsubstituted aryl (e.g., C₆-C₁₀ aryl, C₁₀ aryl, or                phenyl), or unsubstituted heteroaryl (e.g., 5 to 10                membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to                6 membered heteroaryl), and            -   (b) alkyl (e.g., C₁-C₈ alkyl, C₁-C₆ alkyl, or C₁-C₄                alkyl), heteroalkyl (e.g., 2 to 8 membered heteroalkyl,                2 to 6 membered heteroalkyl, or 2 to 4 membered                heteroalkyl), cycloalkyl (e.g., C₃-C₈ cycloalkyl, C₃-C₆                cycloalkyl, or C₅-C₆ cycloalkyl), heterocycloalkyl                (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered                heterocycloalkyl, or 5 to 6 membered heterocycloalkyl),                aryl (e.g., C₆-C₁₀ aryl, C₁₀ aryl, or phenyl),                heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9                membered heteroaryl, or 5 to 6 membered heteroaryl),                substituted with at least one substituent selected from:                oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂, —CHBr₂,                —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH,                —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂,                —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H,                —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃, —OCF₃, —OCBr₃,                —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,                —OCH₂Br, —OCH₂I, —OCH₂F, —N₃, unsubstituted alkyl (e.g.,                C₁-C₈ alkyl, C₁-C₆ alkyl, or C₁-C₄ alkyl), unsubstituted                heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6                membered heteroalkyl, or 2 to 4 membered heteroalkyl),                unsubstituted cycloalkyl (e.g., C₃-C₈ cycloalkyl, C₃-C₆                cycloalkyl, or C₅-C₆ cycloalkyl), unsubstituted                heterocycloalkyl (e.g., 3 to 8 membered                heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5                to 6 membered heterocycloalkyl), unsubstituted aryl                (e.g., C₆-C₁₀ aryl, C₁₀ aryl, or phenyl), or                unsubstituted heteroaryl (e.g., 5 to 10 membered                heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6                membered heteroaryl).

A “size-limited substituent” or “size-limited substituent group,” asused herein, means a group selected from all of the substituentsdescribed above for a “substituent group,” wherein each substituted orunsubstituted alkyl is a substituted or unsubstituted C₁-C₂₀ alkyl, eachsubstituted or unsubstituted heteroalkyl is a substituted orunsubstituted 2 to 20 membered heteroalkyl, each substituted orunsubstituted cycloalkyl is a substituted or unsubstituted C₃-C₈cycloalkyl, each substituted or unsubstituted heterocycloalkyl is asubstituted or unsubstituted 3 to 8 membered heterocycloalkyl, eachsubstituted or unsubstituted aryl is a substituted or unsubstitutedC₆-C₁₀ aryl, and each substituted or unsubstituted heteroaryl is asubstituted or unsubstituted 5 to 10 membered heteroaryl.

A “lower substituent” or “lower substituent group,” as used herein,means a group selected from all of the substituents described above fora “substituent group,” wherein each substituted or unsubstituted alkylis a substituted or unsubstituted C₁-C₈ alkyl, each substituted orunsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8membered heteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₃-C₇ cycloalkyl, each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7membered heterocycloalkyl, each substituted or unsubstituted aryl is asubstituted or unsubstituted phenyl, and each substituted orunsubstituted heteroaryl is a substituted or unsubstituted 5 to 6membered heteroaryl.

In some embodiments, each substituted group described in the compoundsherein is substituted with at least one substituent group. Morespecifically, in some embodiments, each substituted alkyl, substitutedheteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,substituted aryl, substituted heteroaryl, substituted alkylene,substituted heteroalkylene, substituted cycloalkylene, substitutedheterocycloalkylene, substituted arylene, and/or substitutedheteroarylene described in the compounds herein are substituted with atleast one substituent group. In other embodiments, at least one or allof these groups are substituted with at least one size-limitedsubstituent group. In other embodiments, at least one or all of thesegroups are substituted with at least one lower substituent group.

In other embodiments of the compounds herein, each substituted orunsubstituted alkyl may be a substituted or unsubstituted C₁-C₂₀ alkyl,each substituted or unsubstituted heteroalkyl is a substituted orunsubstituted 2 to 20 membered heteroalkyl, each substituted orunsubstituted cycloalkyl is a substituted or unsubstituted C₃-C₈cycloalkyl, each substituted or unsubstituted heterocycloalkyl is asubstituted or unsubstituted 3 to 8 membered heterocycloalkyl, eachsubstituted or unsubstituted aryl is a substituted or unsubstitutedC₆-C₁₀ aryl, and/or each substituted or unsubstituted heteroaryl is asubstituted or unsubstituted 5 to 10 membered heteroaryl. In someembodiments of the compounds herein, each substituted or unsubstitutedalkylene is a substituted or unsubstituted C₁-C₂₀ alkylene, eachsubstituted or unsubstituted heteroalkylene is a substituted orunsubstituted 2 to 20 membered heteroalkylene, each substituted orunsubstituted cycloalkylene is a substituted or unsubstituted C₃-C₅cycloalkylene, each substituted or unsubstituted heterocycloalkylene isa substituted or unsubstituted 3 to 8 membered heterocycloalkylene, eachsubstituted or unsubstituted arylene is a substituted or unsubstitutedC₆-C₁₀ arylene, and/or each substituted or unsubstituted heteroaryleneis a substituted or unsubstituted 5 to 10 membered heteroarylene.

In some embodiments, each substituted or unsubstituted alkyl is asubstituted or unsubstituted C₁-C₈ alkyl, each substituted orunsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8membered heteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₃-C₇ cycloalkyl, each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7membered heterocycloalkyl, each substituted or unsubstituted aryl is asubstituted or unsubstituted phenyl, and/or each substituted orunsubstituted heteroaryl is a substituted or unsubstituted 5 to 6membered heteroaryl. In some embodiments, each substituted orunsubstituted alkylene is a substituted or unsubstituted C₁-C₈ alkylene,each substituted or unsubstituted heteroalkylene is a substituted orunsubstituted 2 to 8 membered heteroalkylene, each substituted orunsubstituted cycloalkylene is a substituted or unsubstituted C₃-C₇cycloalkylene, each substituted or unsubstituted heterocycloalkylene isa substituted or unsubstituted 3 to 7 membered heterocycloalkylene, eachsubstituted or unsubstituted arylene is a substituted or unsubstitutedphenylene, and/or each substituted or unsubstituted heteroarylene is asubstituted or unsubstituted 5 to 6 membered heteroarylene. In someembodiments, the compound (e.g., nucleotide analogue) is a chemicalspecies set forth in the Examples section, claims, embodiments, figures,or tables below.

In embodiments, a substituted or unsubstituted moiety (e.g., substitutedor unsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, substituted or unsubstituted alkylene,substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, and/orsubstituted or unsubstituted heteroarylene) is unsubstituted (e.g., isan unsubstituted alkyl, unsubstituted heteroalkyl, unsubstitutedcycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl,unsubstituted heteroaryl, unsubstituted alkylene, unsubstitutedheteroalkylene, unsubstituted cycloalkylene, unsubstitutedheterocycloalkylene, unsubstituted arylene, and/or unsubstitutedheteroarylene, respectively). In embodiments, a substituted orunsubstituted moiety (e.g., substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl,substituted or unsubstituted alkylene, substituted or unsubstitutedheteroalkylene, substituted or unsubstituted cycloalkylene, substitutedor unsubstituted heterocycloalkylene, substituted or unsubstitutedarylene, and/or substituted or unsubstituted heteroarylene) issubstituted (e.g., is a substituted alkyl, substituted heteroalkyl,substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl,substituted heteroaryl, substituted alkylene, substitutedheteroalkylene, substituted cycloalkylene, substitutedheterocycloalkylene, substituted arylene, and/or substitutedheteroarylene, respectively).

In embodiments, a substituted moiety (e.g., substituted alkyl,substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, substituted aryl, substituted heteroaryl, substitutedalkylene, substituted heteroalkylene, substituted cycloalkylene,substituted heterocycloalkylene, substituted arylene, and/or substitutedheteroarylene) is substituted with at least one substituent group,wherein if the substituted moiety is substituted with a plurality ofsubstituent groups, each substituent group may optionally be different.In embodiments, if the substituted moiety is substituted with aplurality of substituent groups, each substituent group is different.

In embodiments, a substituted moiety (e.g., substituted alkyl,substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, substituted aryl, substituted heteroaryl, substitutedalkylene, substituted heteroalkylene, substituted cycloalkylene,substituted heterocycloalkylene, substituted arylene, and/or substitutedheteroarylene) is substituted with at least one size-limited substituentgroup, wherein if the substituted moiety is substituted with a pluralityof size-limited substituent groups, each size-limited substituent groupmay optionally be different. In embodiments, if the substituted moietyis substituted with a plurality of size-limited substituent groups, eachsize-limited substituent group is different.

In embodiments, a substituted moiety (e.g., substituted alkyl,substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, substituted aryl, substituted heteroaryl, substitutedalkylene, substituted heteroalkylene, substituted cycloalkylene,substituted heterocycloalkylene, substituted arylene, and/or substitutedheteroarylene) is substituted with at least one lower substituent group,wherein if the substituted moiety is substituted with a plurality oflower substituent groups, each lower substituent group may optionally bedifferent. In embodiments, if the substituted moiety is substituted witha plurality of lower substituent groups, each lower substituent group isdifferent.

In embodiments, a substituted moiety (e.g., substituted alkyl,substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, substituted aryl, substituted heteroaryl, substitutedalkylene, substituted heteroalkylene, substituted cycloalkylene,substituted heterocycloalkylene, substituted arylene, and/or substitutedheteroarylene) is substituted with at least one substituent group,size-limited substituent group, or lower substituent group; wherein ifthe substituted moiety is substituted with a plurality of groupsselected from substituent groups, size-limited substituent groups, andlower substituent groups; each substituent group, size-limitedsubstituent group, and/or lower substituent group may optionally bedifferent. In embodiments, if the substituted moiety is substituted witha plurality of groups selected from substituent groups, size-limitedsubstituent groups, and lower substituent groups; each substituentgroup, size-limited substituent group, and/or lower substituent group isdifferent.

Certain compounds of the present disclosure possess asymmetric carbonatoms (optical or chiral centers) or double bonds; the enantiomers,racemates, diastereomers, tautomers, geometric isomers, stereoisometricforms that may be defined, in terms of absolute stereochemistry, as (R)-or (S)- or, as (D)- or (L)- for amino acids, and individual isomers areencompassed within the scope of the present disclosure. The compounds ofthe present disclosure do not include those that are known in art to betoo unstable to synthesize and/or isolate. The present disclosure ismeant to include compounds in racemic and optically pure forms.Optically active (R)- and (S)-, or (D)- and (L)-isomers may be preparedusing chiral synthons or chiral reagents, or resolved using conventionaltechniques. When the compounds described herein contain olefinic bondsor other centers of geometric asymmetry, and unless specified otherwise,it is intended that the compounds include both E and Z geometricisomers.

As used herein, the term “isomers” refers to compounds having the samenumber and kind of atoms, and hence the same molecular weight, butdiffering in respect to the structural arrangement or configuration ofthe atoms.

The term “tautomer,” as used herein, refers to one of two or morestructural isomers which exist in equilibrium and which are readilyconverted from one isomeric form to another.

It will be apparent to one skilled in the art that certain compounds ofthis disclosure may exist in tautomeric forms, all such tautomeric formsof the compounds being within the scope of the disclosure.

Unless otherwise stated, structures depicted herein are also meant toinclude all stereochemical forms of the structure; i.e., the R and Sconfigurations for each asymmetric center. Therefore, singlestereochemical isomers as well as enantiomeric and diastereomericmixtures of the present compounds are within the scope of thedisclosure.

Unless otherwise stated, structures depicted herein are also meant toinclude compounds which differ only in the presence of one or moreisotopically enriched atoms. For example, compounds having the presentstructures except for the replacement of a hydrogen by a deuterium ortritium, or the replacement of a carbon by ¹³C- or ¹⁴C-enriched carbonare within the scope of this disclosure.

The compounds of the present disclosure may also contain unnaturalproportions of atomic isotopes at one or more of the atoms thatconstitute such compounds. For example, the compounds may beradiolabeled with radioactive isotopes, such as for example tritium(³H), iodine-125 (¹²⁵I), or carbon-14 (¹⁴C). All isotopic variations ofthe compounds of the present disclosure, whether radioactive or not, areencompassed within the scope of the present disclosure.

It should be noted that throughout the application that alternatives arewritten in Markush groups, for example, each amino acid position thatcontains more than one possible amino acid. It is specificallycontemplated that each member of the Markush group should be consideredseparately, thereby comprising another embodiment, and the Markush groupis not to be read as a single unit.

“Analog,” “analogue” or “derivative” is used in accordance with itsplain ordinary meaning within Chemistry and Biology and refers to achemical compound that is structurally similar to another compound(i.e., a so-called “reference” compound) but differs in composition,e.g., in the replacement of one atom by an atom of a different element,or in the presence of a particular functional group, or the replacementof one functional group by another functional group, or the absolutestereochemistry of one or more chiral centers of the reference compound.Accordingly, an analog is a compound that is similar or comparable infunction and appearance but not in structure or origin to a referencecompound.

The terms “a” or “an,” as used in herein means one or more. In addition,the phrase “substituted with a[n],” as used herein, means the specifiedgroup may be substituted with one or more of any or all of the namedsubstituents. For example, where a group, such as an alkyl or heteroarylgroup, is “substituted with an unsubstituted C₁-C₂₀ alkyl, orunsubstituted 2 to 20 membered heteroalkyl,” the group may contain oneor more unsubstituted C₁-C₂₀ alkyls, and/or one or more unsubstituted 2to 20 membered heteroalkyls.

Moreover, where a moiety is substituted with an R substituent, the groupmay be referred to as “R-substituted.” Where a moiety is R-substituted,the moiety is substituted with at least one R substituent and each Rsubstituent is optionally different. Where a particular R group ispresent in the description of a chemical genus (such as Formula (I)), aRoman alphabetic symbol may be used to distinguish each appearance ofthat particular R group. For example, where multiple R¹³ substituentsare present, each R¹³ substituent may be distinguished as R^(13A),R^(13B), R^(13C), R^(13D), etc., wherein each of R^(13A), R^(13B),R^(13C), R^(13D), etc. is defined within the scope of the definition ofR¹³ and optionally differently.

As used herein, the term “about” means a range of values including thespecified value, which a person of ordinary skill in the art wouldconsider reasonably similar to the specified value. In embodiments,about means within a standard deviation using measurements generallyacceptable in the art. In embodiments, about means a range extending to+/−10% of the specified value. In embodiments, about includes thespecified value.

A “detectable agent,” “detectable compound,” “detectable label,” or“detectable moiety” is a substance (e.g., element), molecule, orcomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, chemical, magnetic resonance imaging, or other physicalmeans. For example, detectable agents include ¹⁸F, ³²P, ³³P, ⁴⁵Ti, ⁴⁷Sc,⁵²Fe, ⁵⁹Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁷As, ⁸⁶Y, ⁹⁰Y, ⁸⁹Sr, ⁸⁹Zr,⁹⁴Tc, ⁹⁴Tc, ^(99m)Tc, ⁹⁹Mo, ¹⁰⁵Pd, ¹⁰⁵Rh, ¹¹¹Ag, ¹¹¹In, ¹²³I, ¹²⁴I,¹²⁵I, ¹³¹I, ¹⁴²Pr, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁵⁴⁻¹⁵⁸¹Gd, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho,¹⁶⁹Er, ¹⁷⁵Lu, ¹⁷⁷Lu ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ¹⁹⁴Ir, ¹⁹⁸Au, ¹⁹⁹Au, ²¹¹At,²¹¹Pb, ²¹²Bi, ²¹²Pb, ²¹³Bi, ²²³Ra, ²²⁵Ac, Cr, V, Mn, Fe, Co, Ni, Cu, La,Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, ³²P, fluorophore(e.g., fluorescent dyes), modified oligonucleotides (e.g., moietiesdescribed in PCT/US2015/022063, which is incorporated herein byreference), electron-dense reagents, enzymes (e.g., as commonly used inan ELISA), biotin, digoxigenin, paramagnetic molecules, paramagneticnanoparticles, ultrasmall superparamagnetic iron oxide (“USPIO”)nanoparticles, USPIO nanoparticle aggregates, superparamagnetic ironoxide (“SPIO”) nanoparticles, SPIO nanoparticle aggregates,monochrystalline iron oxide nanoparticles, monochrystalline iron oxide,nanoparticle contrast agents, liposomes or other delivery vehiclescontaining Gadolinium chelate (“Gd-chelate”) molecules, Gadolinium,radioisotopes, radionuclides (e.g., carbon-11, nitrogen-13, oxygen-15,fluorine-18, rubidium-82), fluorodeoxyglucose (e.g., fluorine-18labeled), any gamma ray emitting radionuclides, positron-emittingradionuclide, radiolabeled glucose, radiolabeled water, radiolabeledammonia, biocolloids, microbubbles (e.g., including microbubble shellsincluding albumin, galactose, lipid, and/or polymers; microbubble gascore including air, heavy gas(es), perfluorcarbon, nitrogen,octafluoropropane, perflexane lipid microsphere, perflutren, etc.),iodinated contrast agents (e.g., iohexol, iodixanol, ioversol,iopamidol, ioxilan, iopromide, diatrizoate, metrizoate, ioxaglate),barium sulfate, thorium dioxide, gold, gold nanoparticles, goldnanoparticle aggregates, fluorophores, two-photon fluorophores, orhaptens and proteins or other entities which can be made detectable,e.g., by incorporating a radiolabel into a peptide or antibodyspecifically reactive with a target peptide. In embodiments, adetectable moiety is a moiety (e.g., monovalent form) of a detectableagent.

The terms “fluorophore” or “fluorescent agent” or “fluorescent dye” areused interchangeably and refer to a substance, compound, agent (e.g., adetectable agent), or composition (e.g., compound) that can absorb lightat one or more wavelengths and re-emit light at one or more longerwavelengths, relative to the one or more wavelengths of absorbed light.Examples of fluorophores that may be included in the compounds andcompositions described herein include fluorescent proteins, xanthenederivatives (e.g., fluorescein, rhodamine, Oregon green, eosin, or Texasred), cyanine and derivatives (e.g., cyanine, indocarbocyanine,oxacarbocyanine, thiacarbocyanine, or merocyanine), napththalenederivatives (e.g., dansyl or prodan derivatives), coumarin andderivatives, oxadiazole derivatives (e.g., pyridyloxazole,nitrobenzoxadiazole or benzoxadiazole), anthracene derivatives (e.g.,anthraquinones, DRAQ5, DRAQ7, or CyTRAK Orange), pyrene derivatives(e.g., cascade blue and derivatives), oxazine derivatives (e.g., Nilered, Nile blue, cresyl violet, or oxazine 170), acridine derivatives(e.g., proflavin, acridine orange, acridine yellow), arylmethinederivatives (e.g., auramine, crystal violet, or malachite green),tetrapyrrole derivatives (e.g., porphin, phthalocyanine, bilirubin), CFDye™, DRAQ™, CyTRAK™, BODIPY™, Alexa Fluor™, DyLight Fluor™, Atto™,Tracy™ FluoProbes™, Abberior Dyes™, DY™ dyes, MegaStokes Dyes™, SulfoCy™, Seta™ dyes, SeTau™ dyes, Square Dyes™, Quasar™ dyes, Cal Fluor™dyes, SureLight Dyes™, PerCP™ Phycobilisomes™, APC™, APCXL™, RPE™,and/or BPE™. A fluorescent moiety is a radical of a fluorescent agent.The emission from the fluorophores can be detected by any number ofmethods, including but not limited to, fluorescence spectroscopy,fluorescence microscopy, fluorimeters, fluorescent plate readers,infrared scanner analysis, laser scanning confocal microscopy, automatedconfocal nanoscanning, laser spectrophotometers, fluorescent-activatedcell sorters (FACS), image-based analyzers and fluorescent scanners(e.g., gel/membrane scanners). In embodiments, the fluorophore is anaromatic (e.g., polyaromatic) moiety having a conjugated π-electronsystem. In embodiments, the fluorophore is a fluorescent dye moiety,that is, a monovalent fluorophore.

Radioactive substances (e.g., radioisotopes) that may be used as imagingand/or labeling agents in accordance with the embodiments of thedisclosure include, but are not limited to, ¹⁸F, ³²P, ³³P, ⁴⁵Ti, ⁴⁷Sc,⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁷As, ⁸⁶Y, ⁹⁰Y, ⁸⁹Sr, ⁸⁹Zr, ⁹⁴Tc,⁹⁴Tc, ^(99m)Tc, ⁹⁹Mo, ¹⁰⁵Pd, ¹⁰⁵Rh, ¹¹¹Ag, ¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I,¹³¹I, ¹⁴²Pr, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁵⁴⁻¹⁵⁸¹Gd, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho,¹⁶⁹Er, ¹⁷⁵Lu, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ¹⁹⁴Ir, ¹⁹⁸Au, ¹⁹⁹Au, ²¹¹At,²¹¹Pb, ²¹²Bi, ²¹²Pb, ²¹³Bi, ²²³Ra and ²²⁵Ac. Paramagnetic ions that maybe used as additional imaging agents in accordance with the embodimentsof the disclosure include, but are not limited to, ions of transitionand lanthanide metals (e.g., metals having atomic numbers of 21-29, 42,43, 44, or 57-71). These metals include ions of Cr, V, Mn, Fe, Co, Ni,Cu, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu.

Examples of detectable agents include imaging agents, includingfluorescent and luminescent substances, molecules, or compositions,including, but not limited to, a variety of organic or inorganic smallmolecules commonly referred to as “dyes,” “labels,” or “indicators.”Examples include fluorescein, rhodamine, acridine dyes, Alexa dyes, andcyanine dyes. In embodiments, the detectable moiety is a fluorescentmolecule (e.g., acridine dye, cyanine, dye, fluorine dye, oxazine dye,phenanthridine dye, or rhodamine dye). In embodiments, the detectablemoiety is a fluorescent molecule (e.g., acridine dye, cyanine, dye,fluorine dye, oxazine dye, phenanthridine dye, or rhodamine dye). Inembodiments, the detectable moiety is a fluorescent moiety orfluorescent dye moiety. In embodiments, the detectable moiety is afluorescein isothiocyanate moiety, tetramethylrhodamine-5-(and6)-isothiocyanate moiety, Cy2 moiety, Cy3 moiety, Cy5 moiety, Cy7moiety, 4′,6-diamidino-2-phenylindole moiety, Hoechst 33258 moiety,Hoechst 33342 moiety, Hoechst 34580 moiety, propidium-iodide moiety, oracridine orange moiety. In embodiments, the detectable moiety is aIndo-1, Ca saturated moiety, Indo-1 Ca2+ moiety, Cascade Blue BSA pH 7.0moiety, Cascade Blue moiety, LysoTracker Blue moiety, Alexa 405 moiety,LysoSensor Blue pH 5.0 moiety, LysoSensor Blue moiety, DyLight 405moiety, DyLight 350 moiety, BFP (Blue Fluorescent Protein) moiety, Alexa350 moiety, 7-Amino-4-methylcoumarin pH 7.0 moiety, Amino Coumarinmoiety, AMCA conjugate moiety, Coumarin moiety,7-Hydroxy-4-methylcoumarin moiety, 7-Hydroxy-4-methylcoumarin pH 9.0moiety, 6,8-Difluoro-7-hydroxy-4-methylcoumarin pH 9.0 moiety, Hoechst33342 moiety, Pacific Blue moiety, Hoechst 33258 moiety, Hoechst33258-DNA moiety, Pacific Blue antibody conjugate pH 8.0 moiety,PO-PRO-1 moiety, PO-PRO-1-DNA moiety, POPO-1 moiety, POPO-1-DNA moiety,DAPI-DNA moiety, DAPI moiety, Marina Blue moiety, SYTOX Blue-DNA moiety,CFP (Cyan Fluorescent Protein) moiety, eCFP (Enhanced Cyan FluorescentProtein) moiety, 1-Anilinonaphthalene-8-sulfonic acid (1,8-ANS) moiety,Indo-1, Ca free moiety, 1,8-ANS (1-Anilinonaphthalene-8-sulfonic acid)moiety, BO-PRO-1-DNA moiety, BOPRO-1 moiety, BOBO-1-DNA moiety, SYTO45-DNA moiety, evoglow-Ppl moiety, evoglow-Bs1 moiety, evoglow-Bs2moiety, Auramine O moiety, DiO moiety, LysoSensor Green pH 5.0 moiety,Cy 2 moiety, LysoSensor Green moiety, Fura-2, high Ca moiety, Fura-2Ca2+sup> moiety, SYTO 13-DNA moiety, YO-PRO-1-DNA moiety, YOYO-1-DNAmoiety, eGFP (Enhanced Green Fluorescent Protein) moiety, LysoTrackerGreen moiety, GFP (S65T) moiety, BODIPY FL, MeOH moiety, Sapphiremoiety, BODIPY FL conjugate moiety, MitoTracker Green moiety,MitoTracker Green FM, MeOH moiety, Fluorescein 0.1 M NaOH moiety,Calcein pH 9.0 moiety, Fluorescein pH 9.0 moiety, Calcein moiety,Fura-2, no Ca moiety, Fluo-4 moiety, FDA moiety, DTAF moiety,Fluorescein moiety, CFDA moiety, FITC moiety, Alexa Fluor 488hydrazide-water moiety, DyLight 488 moiety, 5-FAM pH 9.0 moiety, Alexa488 moiety, Rhodamine 110 moiety, Rhodamine 110 pH 7.0 moiety, AcridineOrange moiety, BCECF pH 5.5 moiety, PicoGreendsDNA quantitation reagentmoiety, SYBR Green I moiety, Rhodaminen Green pH 7.0 moiety, CyQUANTGR-DNA moiety, NeuroTrace 500/525, green fluorescent Nissl stain-RNAmoiety, DansylCadaverine moiety, Fluoro-Emerald moiety, Nissl moiety,Fluorescein dextran pH 8.0 moiety, Rhodamine Green moiety,5-(and-6)-Carboxy-2′, 7′-dichlorofluorescein pH 9.0 moiety,DansylCadaverine, MeOH moiety, eYFP (Enhanced Yellow FluorescentProtein) moiety, Oregon Green 488 moiety, Fluo-3 moiety, BCECF pH 9.0moiety, SBFI-Na+ moiety, Fluo-3 Ca2+ moiety, Rhodamine 123 MeOH moiety,FlAsH moiety, Calcium Green-1 Ca2+ moiety, Magnesium Green moiety,DM-NERF pH 4.0 moiety, Calcium Green moiety, Citrine moiety, LysoSensorYellow pH 9.0 moiety, TO-PRO-1-DNA moiety, Magnesium Green Mg2+ moiety,Sodium Green Na+ moiety, TOTO-1-DNA moiety, Oregon Green 514 moiety,Oregon Green 514 antibody conjugate pH 8.0 moiety, NBD-X moiety, DM-NERFpH 7.0 moiety, NBD-X, MeOH moiety, CI-NERF pH 6.0 moiety, Alexa 430moiety, CI-NERF pH 2.5 moiety, Lucifer Yellow, CH moiety, LysoSensorYellow pH 3.0 moiety, 6-TET, SE pH 9.0 moiety, Eosin antibody conjugatepH 8.0 moiety, Eosin moiety, 6-Carboxyrhodamine 6G pH 7.0 moiety,6-Carboxyrhodamine 6G, hydrochloride moiety, Bodipy R6G SE moiety,BODIPY R6G MeOH moiety, 6 JOE moiety, Cascade Yellow moiety, mBananamoiety, Alexa 532 moiety, Erythrosin-5-isothiocyanate pH 9.0 moiety,6-HEX, SE pH 9.0 moiety, mOrange moiety, mHoneydew moiety, Cy 3 moiety,Rhodamine B moiety, DiI moiety, 5-TAMRA-MeOH moiety, Alexa 555 moiety,DyLight 549 moiety, BODIPY TMR-X, SE moiety, BODIPY TMR-X MeOH moiety,PO-PRO-3-DNA moiety, PO-PRO-3 moiety, Rhodamine moiety, POPO-3 moiety,Alexa 546 moiety, Calcium Orange Ca2+ moiety, TRITC moiety, CalciumOrange moiety, Rhodaminephalloidin pH 7.0 moiety, MitoTracker Orangemoiety, MitoTracker Orange MeOH moiety, Phycoerythrin moiety, MagnesiumOrange moiety, R-Phycoerythrin pH 7.5 moiety, 5-TAMRA pH 7.0 moiety,5-TAMRA moiety, Rhod-2 moiety, FM 1-43 moiety, Rhod-2 Ca2+ moiety, FM1-43 lipid moiety, LOLO-1-DNA moiety, dTomato moiety, DsRed moiety,Dapoxyl (2-aminoethyl) sulfonamide moiety, Tetramethylrhodamine dextranpH 7.0 moiety, Fluor-Ruby moiety, Resorufin moiety, Resorufin pH 9.0moiety, mTangerine moiety, LysoTracker Red moiety, Lissaminerhodaminemoiety, Cy 3.5 moiety, Rhodamine Red-X antibody conjugate pH 8.0 moiety,Sulforhodamine 101 EtOH moiety, JC-1 pH 8.2 moiety, JC-1 moiety,mStrawberry moiety, MitoTracker Red moiety, MitoTracker Red, MeOHmoiety, X-Rhod-1 Ca2+ moiety, Alexa 568 moiety, 5-ROX pH 7.0 moiety,5-ROX (5-Carboxy-X-rhodamine, triethylammonium salt) moiety,BO-PRO-3-DNA moiety, BOPRO-3 moiety, BOBO-3-DNA moiety, Ethidium Bromidemoiety, ReAsH moiety, Calcium Crimson moiety, Calcium Crimson Ca2+moiety, mRFP moiety, mCherry moiety, HcRed moiety, DyLight 594 moiety,Ethidium homodimer-1-DNA moiety, Ethidiumhomodimer moiety, PropidiumIodide moiety, SYPRO Ruby moiety, Propidium Iodide-DNA moiety, Alexa 594moiety, BODIPY TR-X, SE moiety, BODIPY TR-X, MeOH moiety, BODIPY TR-Xphallacidin pH 7.0 moiety, Alexa Fluor 610 R-phycoerythrin streptavidinpH 7.2 moiety, YO-PRO-3-DNA moiety, Di-8 ANEPPS moiety,Di-8-ANEPPS-lipid moiety, YOYO-3-DNA moiety, Nile Red-lipid moiety, NileRed moiety, DyLight 633 moiety, mPlum moiety, TO-PRO-3-DNA moiety, DDAOpH 9.0 moiety, Fura Red high Ca moiety, Allophycocyanin pH 7.5 moiety,APC (allophycocyanin) moiety, Nile Blue, EtOH moiety, TOTO-3-DNA moiety,Cy 5 moiety, BODIPY 650/665-X, MeOH moiety, Alexa Fluor 647R-phycoerythrin streptavidin pH 7.2 moiety, DyLight 649 moiety, Alexa647 moiety, Fura Red Ca2+ moiety, Atto 647 moiety, Fura Red, low Camoiety, Carboxynaphthofluorescein pH 10.0 moiety, Alexa 660 moiety, Cy5.5 moiety, Alexa 680 moiety, DyLight 680 moiety, Alexa 700 moiety, FM4-64, 2% CHAPS moiety, or FM 4-64 moiety. In embodiments, the dectablemoiety is a moiety of 1,1-Diethyl-4,4-carbocyanine iodide,1,2-Diphenylacetylene, 1,4-Diphenylbutadiene, 1,4-Diphenylbutadiyne,1,6-Diphenylhexatriene, 1,6-Diphenylhexatriene,1-anilinonaphthalene-8-sulfonic acid, 2,7-Dichlorofluorescein,2,5-DIPHENYLOXAZOLE, 2-Di-1-ASP, 2-dodecylresorufin,2-Methylbenzoxazole, 3,3-Diethylthiadicarbocyanine iodide,4-Dimethylamino-4-Nitrostilbene, 5(6)-Carboxyfluorescein,5(6)-Carboxynaphtofluorescein, 5(6)-Carboxytetramethylrhodamine B,5-(and-6)-carboxy-2′,7′-dichlorofluorescein,5-(and-6)-carboxy-2,7-dichlorofluorescein, 5-(N-hexadecanoyl)aminoeosin,5-(N-hexadecanoyl)aminoeosin, 5-chloromethylfluorescein, 5-FAM, 5-ROX,5-TAMRA, 5-TAMRA, 6,8-difluoro-7-hydroxy-4-methylcoumarin,6,8-difluoro-7-hydroxy-4-methylcoumarin, 6-carboxyrhodamine 6G, 6-HEX,6-JOE, 6-JOE, 6-TET, 7-aminoactinomycin D,7-Benzylamino-4-Nitrobenz-2-Oxa-1,3-Diazole, 7-Methoxycoumarin-4-AceticAcid, 8-Benzyloxy-5,7-diphenylquinoline,8-Benzyloxy-5,7-diphenylquinoline, 9,10-Bis(Phenylethynyl)Anthracene,9,10-Diphenylanthracene, 9-METHYLCARBAZOLE, (CS)2Ir(μ-Cl)2Ir(CS)2, AAA,Acridine Orange, Acridine Orange, Acridine Yellow, Acridine Yellow,Adams Apple Red 680, Adirondack Green 520, Alexa Fluor 350, Alexa Fluor405, Alexa Fluor 430, Alexa Fluor 430, Alexa Fluor 480, Alexa Fluor 488,Alexa Fluor 488, Alexa Fluor 488 hydrazide, Alexa Fluor 500, Alexa Fluor514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 546, Alexa Fluor 555,Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 594,Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 610-R-PE, Alexa Fluor 633,Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 647, Alexa Fluor 647-R-PE,Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 680-APC, Alexa Fluor680-R-PE, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790,Allophycocyanin, AmCyan1, Aminomethylcoumarin, Amplex Gold (product),Amplex Red Reagent, Amplex UltraRed, Anthracene, APC, APC-Seta-750,AsRed2, ATTO 390, ATTO 425, ATTO 430LS, ATTO 465, ATTO 488, ATTO 490LS,ATTO 495, ATTO 514, ATTO 520, ATTO 532, ATTO 550, ATTO 565, ATTO 590,ATTO 594, ATTO 610, ATTO 620, ATTO 633, ATTO 635, ATTO 647, ATTO 647N,ATTO 655, ATTO 665, ATTO 680, ATTO 700, ATTO 725, ATTO 740, ATTO Oxa12,ATTO Rho3B, ATTO Rho6G, ATTO Rho 11, ATTO Rho12, ATTO Rho13, ATTO Rho14,ATTO Rho101, ATTO Thio12, Auramine O, Azami Green, Azami Greenmonomeric, B-phycoerythrin, BCECF, BCECF, Bex1, Biphenyl, Birch Yellow580, Blue-green algae, BO-PRO-1, BO-PRO-3, BOBO-1, BOBO-3, BODIPY 630650-X, BODIPY 650/665-X, BODIPY FL, BODIPY FL, BODIPY R6G, BODIPY TMR-X,BODIPY TR-X, BODIPY TR-X Ph 7.0, BODIPY TR-X phallacidin, BODIPY-DiMe,BODIPY-Phenyl, BODIPY-TMSCC, C3-Indocyanine, C3-Indocyanine,C3-Oxacyanine, C3-Thiacyanine Dye (EtOH), C3-Thiacyanine Dye (PrOH),C5-Indocyanine, C5-Oxacyanine, C5-Thiacyanine, C7-Indocyanine,C7-Oxacyanine, C545T, C-Phycocyanin, Calcein, Calcein red-orange,Calcium Crimson, Calcium Green-1, Calcium Orange, Calcofluor white 2MR,Carboxy SNARF-1 pH 6.0, Carboxy SNARF-1 pH 9.0,Carboxynaphthofluorescein, Cascade Blue, Cascade Yellow, Catskill Green540, CBQCA, CellMask Orange, CellTrace BODIPY TR methyl ester, CellTracecalcein violet, CellTrace™ Far Red, CellTracker Blue, CellTracker RedCMTPX, CellTracker Violet BMQC, CF405M, CF405S, CF488A, CF543, CF555,CFP, CFSE, CF™ 350, CF™ 485, Chlorophyll A, Chlorophyll B, Chromeo 488,Chromeo 494, Chromeo 505, Chromeo 546, Chromeo 642, Citrine, Citrine,ClOH butoxy aza-BODIPY, ClOH C12 aza-BODIPY, CM-H2DCFDA, Coumarin 1,Coumarin 6, Coumarin 6, Coumarin 30, Coumarin 314, Coumarin 334,Coumarin 343, Coumarine 545T, Cresyl Violet Perchlorate, CryptoLightCF1, CryptoLight CF2, CryptoLight CF3, CryptoLight CF4, CryptoLight CF5,CryptoLight CF6, Crystal Violet, Cumarin153, Cy2, Cy3, Cy3, Cy3.5, Cy3B,Cy3B, Cy3Cy5 ET, Cy5, Cy5, Cy5.5, Cy7, Cyanine3 NHS ester, Cyanine5carboxylic acid, Cyanine5 NHS ester, Cyclotella meneghiniana Kutzing,CypHer5, CypHer5 pH 9.15, CyQUANT GR, CyTrak Orange, Dabcyl SE, DAF-FM,DAMC (Weiss), dansyl cadaverine, Dansyl Glycine (Dioxane), DAPI, DAPI,DAPI, DAPI, DAPI (DMSO), DAPI (H2O), Dapoxyl (2-aminoethyl)sulfonamide,DCI, DCM, DCM, DCM (acetonitrile), DCM (MeOH), DDAO, Deep Purple,di-8-ANEPPS, DiA, Dichlorotris(1,10-phenanthroline) ruthenium(II),DiClOH C12 aza-BODIPY, DiClOHbutoxy aza-BODIPY, DiD, DiI, DiIC18(3),DiO, DiR, Diversa Cyan-FP, Diversa Green-FP, DM-NERF pH 4.0, DOCI,Doxorubicin, DPP pH-Probe 590-7.5, DPP pH-Probe 590-9.0, DPP pH-Probe590-11.0, DPP pH-Probe 590-11.0, Dragon Green, DRAQ5, DsRed, DsRed,DsRed, DsRed-Express, DsRed-Express2, DsRed-Express T1, dTomato,DY-350XL, DY-480, DY-480XL MegaStokes, DY-485, DY-485XL MegaStokes,DY-490, DY-490XL MegaStokes, DY-500, DY-500XL MegaStokes, DY-520,DY-520XL MegaStokes, DY-547, DY-549P1, DY-549P1, DY-554, DY-555, DY-557,DY-557, DY-590, DY-590, DY-615, DY-630, DY-631, DY-633, DY-635, DY-636,DY-647, DY-649P1, DY-649P1, DY-650, DY-651, DY-656, DY-673, DY-675,DY-676, DY-680, DY-681, DY-700, DY-701, DY-730, DY-731, DY-750, DY-751,DY-776, DY-782, Dye-28, Dye-33, Dye-45, Dye-304, Dye-1041, DyLight 488,DyLight 549, DyLight 594, DyLight 633, DyLight 649, DyLight 680,E2-Crimson, E2-Orange, E2-Red/Green, EBFP, ECF, ECFP, ECL Plus, eGFP,ELF 97, Emerald, Envy Green, Eosin, Eosin Y, epicocconone, EqFP611,Erythrosin-5-isothiocyanate, Ethidium bromide, ethidium homodimer-1,Ethyl Eosin, Ethyl Eosin, Ethyl Nile Blue A,Ethyl-p-Dimethylaminobenzoate, Ethyl-p-Dimethylaminobenzoate, Eu203nanoparticles, Eu (Soini), Eu(tta)3DEADIT, EvaGreen, EVOblue-30, EYFP,FAD, FITC, FITC, FlAsH (Adams), Flash Red EX, FlAsH-CCPGCC,FlAsH-CCXXCC, Fluo-3, Fluo-4, Fluo-5F, Fluorescein, Fluorescein 0.1NaOH, Fluorescein-Dibase, fluoro-emerald, Fluorol 5G, FluoSpheres blue,FluoSpheres crimson, FluoSpheres dark red, FluoSpheres orange,FluoSpheres red, FluoSpheres yellow-green, FM4-64 in CTC, FM4-64 in SDS,FM 1-43, FM 4-64, Fort Orange 600, Fura Red, Fura Red Ca free, fura-2,Fura-2 Ca free, Gadodiamide, Gd-Dtpa-Bma, Gadodiamide, Gd-Dtpa-Bma,GelGreen™, GelRed™, H9-40, HcRed1, Hemo Red 720, HiLyte Fluor 488,HiLyte Fluor 555, HiLyte Fluor 647, HiLyte Fluor 680, HiLyte Fluor 750,HiLyte Plus 555, HiLyte Plus 647, HiLyte Plus 750, HmGFP, Hoechst 33258,Hoechst 33342, Hoechst-33258, Hoechst-33258, Hops Yellow 560, HPTS,HPTS, HPTS, HPTS, HPTS, indo-1, Indo-1 Ca free, Ir(Cn)2(acac),Ir(Cs)2(acac), IR-775 chloride, IR-806, Ir-OEP-CO-Cl, IRDye® 650 Alkyne,IRDye® 650 Azide, IRDye® 650 Carboxylate, IRDye® 650 DBCO, TRDye® 650Maleimide, TRDye® 650 NHS Ester, IRDye® 680LT Carboxylate, IRDye® 680LTMaleimide, IRDye® 680LT NHS Ester, IRDye® 680RD Alkyne, IRDye® 680RDAzide, IRDye® 680RD Carboxylate, IRDye® 680RD DBCO, IRDye® 680RDMaleimide, IRDye® 680RD NHS Ester, TRDye® 700 phosphoramidite, IRDye®700DX, IRDye® 700DX, IRDye® 700DX Carboxylate, IRDye® 700DX NHS Ester,IRDye® 750 Carboxylate, IRDye® 750 Maleimide, IRDye® 750 NHS Ester,IRDye® 800 phosphoramidite, IRDye® 800CW, IRDye® 800CW Alkyne, TRDye®800CW Azide, IRDye® 800CW Carboxylate, IRDye® 800CW DBCO, IRDye® 800CWMaleimide, IRDye® 800CW NHS Ester, IRDye® 800RS, IRDye® 800RSCarboxylate, IRDye® 800RS NHS Ester, IRDye® QC-1 Carboxylate, TRDye®QC-1 NHS Ester, Isochrysis galbana-Parke, JC-1, JC-1, JOJO-1, JonamacRed Evitag T2, Kaede Green, Kaede Red, kusabira orange, Lake Placid 490,LDS 751, Lissamine Rhodamine (Weiss), LOLO-1, lucifer yellow CH, LuciferYellow CH, lucifer yellow CH, Lucifer Yellow CH Dilitium salt, LumioGreen, Lumio Red, Lumogen F Orange, Lumogen Red F300, Lumogen Red F300,LysoSensor Blue DND-192, LysoSensor Green DND-153, LysoSensor GreenDND-153, LysoSensor Yellow/Blue DND-160 pH 3, LysoSensor YellowBlueDND-160, LysoTracker Blue DND-22, LysoTracker Blue DND-22, LysoTrackerGreen DND-26, LysoTracker Red DND-99, LysoTracker Yellow HCK-123, MacounRed Evitag T2, Macrolex Fluorescence Red G, Macrolex Fluorescence Yellow10GN, Macrolex Fluorescence Yellow 10GN, Magnesium Green, MagnesiumOctaethylporphyrin, Magnesium Orange, Magnesium Phthalocyanine,Magnesium Phthalocyanine, Magnesium Tetramesitylporphyrin, MagnesiumTetraphenylporphyrin, malachite green isothiocyanate, Maple Red-Orange620, Marina Blue, mBanana, mBBr, mCherry, Merocyanine 540, Methyl green,Methyl green, Methyl green, Methylene Blue, Methylene Blue, mHoneyDew,MitoTracker Deep Red 633, MitoTracker Green FM, MitoTracker OrangeCMTMRos, MitoTracker Red CMXRos, monobromobimane, Monochlorobimane,Monoraphidium, mOrange, mOrange2, mPlum, mRaspberry, mRFP, mRFP1,mRFP1.2 (Wang), mStrawberry (Shaner), mTangerine (Shaner),N,N-Bis(2,4,6-trimethylphenyl)-3,4:9,10-perylenebis(dicarboximide),NADH, Naphthalene, Naphthalene, Naphthofluorescein, Naphthofluorescein,NBD-X, NeuroTrace 500525, Nilblau perchlorate, nile blue, Nile Blue,Nile Blue (EtOH), nile red, Nile Red, Nile Red, Nile red, Nileblue A,NIR1, NIR2, NIR3, NIR4, NIR820, Octaethylporphyrin, OH butoxyaza-BODIPY, OHC12 aza-BODIPY, Orange Fluorescent Protein, Oregon Green488, Oregon Green 488 DUPE, Oregon Green 514, Oxazin1, Oxazin 750,Oxazine 1, Oxazine 170, P4-3, P-Quaterphenyl, P-Terphenyl, PA-GFP(post-activation), PA-GFP (pre-activation), Pacific Orange,Palladium(II) meso-tetraphenyl-tetrabenzoporphyrin, PdOEPK, PdTFPP,PerCP-Cy5.5, Perylene, Perylene, Perylene bisimide pH-Probe 550-5.0,Perylene bisimide pH-Probe 550-5.5, Perylene bisimide pH-Probe 550-6.5,Perylene Green pH-Probe 720-5.5, Perylene Green Tag pH-Probe 720-6.0,Perylene Orange pH-Probe 550-2.0, Perylene Orange Tag 550, Perylene RedpH-Probe 600-5.5, Perylenediimid, Perylne Green pH-Probe 740-5.5,Phenol, Phenylalanine, pHrodo, succinimidyl ester, Phthalocyanine,PicoGreen dsDNA quantitation reagent, Pinacyanol-Iodide, Piroxicam,Platinum(II) tetraphenyltetrabenzoporphyrin, Plum Purple, PO-PRO-1,PO-PRO-3, POPO-1, POPO-3, POPOP, Porphin, PPO, Proflavin,PromoFluor-350, PromoFluor-405, PromoFluor-415, PromoFluor-488,PromoFluor-488 Premium, PromoFluor-488LSS, PromoFluor-500LSS,PromoFluor-505, PromoFluor-510LSS, PromoFluor-514LSS, PromoFluor-520LSS,PromoFluor-532, PromoFluor-546, PromoFluor-555, PromoFluor-590,PromoFluor-610, PromoFluor-633, PromoFluor-647, PromoFluor-670,PromoFluor-680, PromoFluor-700, PromoFluor-750, PromoFluor-770,PromoFluor-780, PromoFluor-840, propidium iodide, Protoporphyrin IX,PTIR475/UF, PTIR545/UF, PtOEP, PtOEPK, PtTFPP, Pyrene, QD525, QD565,QD585, QD605, QD655, QD705, QD800, QD903, QD PbS 950, QDot 525, QDot545, QDot 565, Qdot 585, Qdot 605, Qdot 625, Qdot 655, Qdot 705, Qdot800, QpyMe2, QSY 7, QSY 7, QSY 9, QSY 21, QSY 35, quinine, QuinineSulfate, Quinine sulfate, R-phycoerythrin, R-phycoerythrin,ReAsH-CCPGCC, ReAsH-CCXXCC, Red Beads (Weiss), Redmond Red, Resorufin,resorufin, rhod-2, Rhodamin 700 perchlorate, rhodamine, Rhodamine 6G,Rhodamine 6G, Rhodamine 101, rhodamine 110, Rhodamine 123, rhodamine123, Rhodamine B, Rhodamine B, Rhodamine Green, Rhodamine pH-Probe585-7.0, Rhodamine pH-Probe 585-7.5, Rhodamine phalloidin, RhodamineRed-X, Rhodamine Red-X, Rhodamine Tag pH-Probe 585-7.0, Rhodol Green,Riboflavin, Rose Bengal, Sapphire, SBFI, SBFI Zero Na, Scenedesmus sp.,SensiLight PBXL-1, SensiLight PBXL-3, Seta 633-NHS, Seta-633-NHS,SeTau-380-NHS, SeTau-647-NHS, Snake-Eye Red 900, SNIR1, SNIR2, SNIR3,SNIR4, Sodium Green, Solophenyl flavine 7GFE 500, Spectrum Aqua,Spectrum Blue, Spectrum FRed, Spectrum Gold, Spectrum Green, SpectrumOrange, Spectrum Red, Squarylium dye III, Stains All, Stilben derivate,Stilbene, Styryl8 perchlorate, Sulfo-Cyanine3 carboxylic acid,Sulfo-Cyanine3 carboxylic acid, Sulfo-Cyanine3 NHS ester, Sulfo-Cyanine5carboxylic acid, Sulforhodamine 101, sulforhodamine 101, SulforhodamineB, Sulforhodamine G, Suncoast Yellow, SuperGlo BFP, SuperGlo GFP, SurfGreen EX, SYBR Gold nucleic acid gel stain, SYBR Green I, SYPRO Ruby,SYTO 9, SYTO 11, SYTO 13, SYTO 16, SYTO 17, SYTO 45, SYTO 59, SYTO 60,SYTO 61, SYTO 62, SYTO 82, SYTO RNASelect, SYTO RNASelect, SYTOX Blue,SYTOX Green, SYTOX Orange, SYTOX Red, T-Sapphire, Tb (Soini), tCO,tdTomato, Terrylen, Terrylendiimid, testdye, Tetra-t-Butylazaporphine,Tetra-t-Butylnaphthalocyanine, Tetracen,Tetrakis(o-Aminophenyl)Porphyrin, Tetramesitylporphyrin,Tetramethylrhodamine, tetramethylrhodamine, Tetraphenylporphyrin,Tetraphenylporphyrin, Texas Red, Texas Red DUPE, Texas Red-X,ThiolTracker Violet, Thionin acetate, TMRE, TO-PRO-1, TO-PRO-3, Toluene,Topaz (Tsienl998), TOTO-1, TOTO-3, Tris(2,2-Bipyridyl)Ruthenium(II)chloride, Tri s(4,4-diphenyl-2,2-bipyridine) ruthenium(II) chloride,Tris(4,7-diphenyl-1,10-phenanthroline) ruthenium(II) TMS, TRITC (Weiss),TRITC Dextran (Weiss), Tryptophan, Tyrosine, Vex1, Vybrant DyeCycleGreen stain, Vybrant DyeCycle Orange stain, Vybrant DyeCycle Violetstain, WEGFP (post-activation), WellRED D2, WellRED D3, WellRED D4,WtGFP, WtGFP (Tsienl998), X-rhod-1, Yakima Yellow, YFP, YO-PRO-1,YO-PRO-3, YOYO-1, YoYo-1, YoYo-1 dsDNA, YoYo-1 ssDNA, YOYO-3, ZincOctaethylporphyrin, Zinc Phthalocyanine, Zinc Tetramesitylporphyrin,Zinc Tetraphenylporphyrin, ZsGreenl, or ZsYellowl. In embodiments, R⁴ isa monovalent moiety of a compound described within this paragraph.

In embodiments, the detectable moiety is a moiety of a derivative of oneof the detectable moieties described immediately above, wherein thederivative differs from one of the detectable moieties immediately aboveby a modification resulting from the conjugation of the detectablemoiety to a compound described herein.

In embodiments, the detectable label is a fluorescent dye. Inembodiments, the detectable label is a fluorescent dye capable ofexchanging energy with another fluorescent dye (e.g., fluorescenceresonance energy transfer (FRET) chromophores).

The term “cyanine” or “cyanine moiety” as described herein refers to adetectable moiety containing two nitrogen groups separated by apolymethine chain. In embodiments, the cyanine moiety has 3 methinestructures (i.e., cyanine 3 or Cy3). In embodiments, the cyanine moietyhas 5 methine structures (i.e., cyanine 5 or Cy5). In embodiments, thecyanine moiety has 7 methine structures (i.e., cyanine 7 or Cy7).

Descriptions of compounds (e.g., nucleotide analogues) of the presentdisclosure are limited by principles of chemical bonding known to thoseskilled in the art. Accordingly, where a group may be substituted by oneor more of a number of substituents, such substitutions are selected soas to comply with principles of chemical bonding and to give compoundswhich are not inherently unstable and/or would be known to one ofordinary skill in the art as likely to be unstable under ambientconditions, such as aqueous, neutral, and several known physiologicalconditions. For example, a heterocycloalkyl or heteroaryl is attached tothe remainder of the molecule via a ring heteroatom in compliance withprinciples of chemical bonding known to those skilled in the art therebyavoiding inherently unstable compounds.

The term “pharmaceutically acceptable salts” is meant to include saltsof the active compounds that are prepared with relatively nontoxic acidsor bases, depending on the particular substituents found on thecompounds described herein. When compounds of the present inventioncontain relatively acidic functionalities, base addition salts can beobtained by contacting the neutral form of such compounds with asufficient amount of the desired base, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable base additionsalts include sodium, potassium, calcium, ammonium, organic amino, ormagnesium salt, or a similar salt. When compounds of the presentinvention contain relatively basic functionalities, acid addition saltscan be obtained by contacting the neutral form of such compounds with asufficient amount of the desired acid, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable acid additionsalts include those derived from inorganic acids like hydrochloric,hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, maleic, malonic, benzoic, succinic,suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, oxalic, methanesulfonic, and thelike. Also included are salts of amino acids such as arginate and thelike, and salts of organic acids like glucuronic or galactunoric acidsand the like (see, for example, Berge et al., “Pharmaceutical Salts”,Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specificcompounds of the present invention contain both basic and acidicfunctionalities that allow the compounds to be converted into eitherbase or acid addition salts.

As used herein, the term “salt” refers to acid or base salts of thecompounds described herein. Thus, the compounds of the present inventionmay exist as salts, such as with pharmaceutically acceptable acids. Thepresent invention includes such salts. Non-limiting examples of suchsalts include hydrochlorides, hydrobromides, phosphates, sulfates,methanesulfonates, nitrates, maleates, acetates, citrates, fumarates,proprionates, tartrates (e.g., (+)-tartrates, (−)-tartrates, or mixturesthereof including racemic mixtures), succinates, benzoates, and saltswith amino acids such as glutamic acid, and quaternary ammonium salts(e.g., methyl iodide, ethyl iodide, and the like). These salts may beprepared by methods known to those skilled in the art. Illustrativeexamples of acceptable salts are mineral acid (hydrochloric acid,hydrobromic acid, phosphoric acid, and the like) salts, organic acid(acetic acid, propionic acid, glutamic acid, citric acid and the like)salts, quaternary ammonium (methyl iodide, ethyl iodide, and the like)salts. In embodiments, compounds may be presented with a positivecharge, and it is understood an appropriate counter-ion (e.g., chlorideion, fluoride ion, or acetate ion) may also be present, though notexplicitly shown. Likewise, for compounds having a negative charge(e.g.,

it is understood an appropriate counter-ion (e.g., a proton, sodium ion,potassium ion, or ammonium ion) may also be present, though notexplicitly shown. The protonation state of the compound (e.g., acompound described herein) depends on the local environment (i.e., thepH of the environment), therefore, in embodiments, the compound may bedescribed as having a moiety in a protonated state (e.g.,

or an ionic state (e.g.,

and it is understood these are interchangeable. In embodiments, thecounter-ion is represented by the symbol M (e.g., M⁺ or M⁻).

The neutral forms of the compounds are preferably regenerated bycontacting the salt with a base or acid and isolating the parentcompound in the conventional manner. The parent form of the compound maydiffer from the various salt forms in certain physical properties, suchas solubility in polar solvents.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are encompassedwithin the scope of the present invention. Certain compounds of thepresent invention may exist in multiple crystalline or amorphous forms.In general, all physical forms are equivalent for the uses contemplatedby the present invention and are intended to be within the scope of thepresent invention.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues,wherein the polymer may optionally be conjugated to a moiety that doesnot consist of amino acids. The terms apply to amino acid polymers inwhich one or more amino acid residue is an artificial chemical mimeticof a corresponding naturally occurring amino acid, as well as tonaturally occurring amino acid polymers and non-naturally occurringamino acid polymer.

A polypeptide, or a cell is “recombinant” when it is artificial orengineered, or derived from or contains an artificial or engineeredprotein or nucleic acid (e.g., non-natural or not wild type). Forexample, a polynucleotide that is inserted into a vector or any otherheterologous location, e.g., in a genome of a recombinant organism, suchthat it is not associated with nucleotide sequences that normally flankthe polynucleotide as it is found in nature is a recombinantpolynucleotide. A protein expressed in vitro or in vivo from arecombinant polynucleotide is an example of a recombinant polypeptide.Likewise, a polynucleotide sequence that does not appear in nature, forexample a variant of a naturally occurring gene, is recombinant.

“Hybridize” shall mean the annealing of one single-stranded nucleic acid(such as a primer) to another nucleic acid based on the well-understoodprinciple of sequence complementarity. In an embodiment the othernucleic acid is a single-stranded nucleic acid. The propensity forhybridization between nucleic acids depends on the temperature and ionicstrength of their milieu, the length of the nucleic acids and the degreeof complementarity. The effect of these parameters on hybridization isdescribed in, for example, Sambrook J., Fritsch E. F., Maniatis T.,Molecular cloning: a laboratory manual, Cold Spring Harbor LaboratoryPress, New York (1989). As used herein, hybridization of a primer, or ofa DNA extension product, respectively, is extendable by creation of aphosphodiester bond with an available nucleotide or nucleotide analoguecapable of forming a phosphodiester bond, therewith. Those skilled inthe art understand how to estimate and adjust the stringency ofhybridization conditions such that sequences having at least a desiredlevel of complementarity will stably hybridize, while those having lowercomplementarity will not. As used herein, the term “stringent condition”refers to condition(s) under which a polynucleotide probe or primer willhybridize preferentially to its target sequence, and to a lesser extentto, or not at all to, other sequences. In some embodiments nucleicacids, or portions thereof, that are configured to specificallyhybridize are often about 80% or more, 81% or more, 82% or more, 83% ormore, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more,89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% ormore, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more or100% complementary to each other over a contiguous portion of nucleicacid sequence. A specific hybridization discriminates over non-specifichybridization interactions (e.g., two nucleic acids that a notconfigured to specifically hybridize, e.g., two nucleic acids that are80% or less, 70% or less, 60% or less or 50% or less complementary) byabout 2-fold or more, often about 10-fold or more, and sometimes about100-fold or more, 1000-fold or more, 10,000-fold or more, 100,000-foldor more, or 1,000,000-fold or more. Two nucleic acid strands that arehybridized to each other can form a duplex which comprises adouble-stranded portion of nucleic acid.

“Contacting” is used in accordance with its plain ordinary meaning andrefers to the process of allowing at least two distinct species (e.g.,chemical compounds including biomolecules or cells) to becomesufficiently proximal to react, interact or physically touch. It shouldbe appreciated, however, that the resulting reaction product can beproduced directly from a reaction between the added reagents or from anintermediate from one or more of the added reagents that can be producedin the reaction mixture. The term “contacting” may include allowing twospecies to react, interact, or physically touch, wherein the two speciesmay be a compound as described herein and a protein or enzyme. In someembodiments contacting includes allowing a compound described herein tointeract with a protein or enzyme that is involved in a signalingpathway.

The term “streptavidin” refers to a tetrameric protein (includinghomologs, isoforms, and functional fragments thereof) capable of bindingbiotin. The term includes any recombinant or naturally-occurring form ofstreptavidin variants thereof that maintain streptavidin activity (e.g.,within at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% activitycompared to wildtype streptavidin).

The term “expression” includes any step involved in the production ofthe polypeptide including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion. Expression can be detected usingconventional techniques for detecting protein (e.g., ELISA, Westernblotting, flow cytometry, immunofluorescence, immunohistochemistry,etc.).

An “effective amount” is an amount sufficient for a compound toaccomplish a stated purpose relative to the absence of the compound(e.g., achieve the effect for which it is administered, treat a disease,reduce enzyme activity, increase enzyme activity, reduce a signalingpathway, or reduce one or more symptoms of a disease or condition). An“activity decreasing amount,” as used herein, refers to an amount ofantagonist required to decrease the activity of an enzyme relative tothe absence of the antagonist. A “function disrupting amount,” as usedherein, refers to the amount of antagonist required to disrupt thefunction of an enzyme or protein relative to the absence of theantagonist.

“Control” or “control experiment” is used in accordance with its plainordinary meaning and refers to an experiment in which the subjects orreagents of the experiment are treated as in a parallel experimentexcept for omission of a procedure, reagent, or variable of theexperiment. In some instances, the control is used as a standard ofcomparison in evaluating experimental effects. In some embodiments, acontrol is the measurement of the activity of a protein in the absenceof a compound as described herein (including embodiments and examples).

The term “modulate” is used in accordance with its plain ordinarymeaning and refers to the act of changing or varying one or moreproperties. “Modulation” refers to the process of changing or varyingone or more properties. For example, as applied to the effects of amodulator on a target protein, to modulate means to change by increasingor decreasing a property or function of the target molecule or theamount of the target molecule.

“Nucleic acid” refers to nucleotides (e.g., deoxyribonucleotides orribonucleotides) and polymers thereof in either single-, double- ormultiple-stranded form, or complements thereof, or nucleosides (e.g.,deoxyribonucleosides or ribonucleosides). In embodiments, “nucleic acid”does not include nucleosides. The terms “polynucleotide,”“oligonucleotide,” “oligo” or the like refer, in the usual and customarysense, to a linear sequence of nucleotides. Oligonucleotides aretypically from about 5, 6, 7, 8, 9, 10, 12, 15, 25, 30, 40, 50 or morenucleotides in length, up to about 100 nucleotides in length. Nucleicacids and polynucleotides are polymers of any length, including longerlengths, e.g., 200, 300, 500, 1000, 2000, 3000, 5000, 7000, 10,000, etc.In certain embodiments the nucleic acids herein contain phosphodiesterbonds. In other embodiments, nucleic acid analogs are included that mayhave alternate backbones, comprising, e.g., phosphoramidate,phosphorothioate, phosphorodithioate, or O-methylphosphoroamiditelinkages (see, Eckstein, Oligonucleotides and Analogues: A PracticalApproach, Oxford University Press); and peptide nucleic acid backbonesand linkages. Other analog nucleic acids include those with positivebackbones; non-ionic backbones, and non-ribose backbones, includingthose described in U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters6 and 7, ASC Symposium Series 580, Carbohydrate Modifications inAntisense Research, Sanghui & Cook, eds. Nucleic acids containing one ormore carbocyclic sugars are also included within one definition ofnucleic acids. Modifications of the ribose-phosphate backbone may bedone for a variety of reasons, e.g., to increase the stability andhalf-life of such molecules in physiological environments or as probeson a biochip. Mixtures of naturally occurring nucleic acids and analogscan be made; alternatively, mixtures of different nucleic acid analogs,and mixtures of naturally occurring nucleic acids and analogs may bemade. A residue of a nucleic acid, as referred to herein, is a monomerof the nucleic acid (e.g., a nucleotide). The term “nucleoside” refers,in the usual and customary sense, to a glycosylamine including anucleobase and a five-carbon sugar (ribose or deoxyribose). Non-limitingexamples of nucleosides include cytidine, uridine, adenosine, guanosine,thymidine and inosine. Nucleosides may be modified at the base and/orthe sugar. The term “nucleotide” refers, in the usual and customarysense, to a single unit of a polynucleotide, i.e., a monomer.Nucleotides can be ribonucleotides, deoxyribonucleotides, or modifiedversions thereof. Examples of polynucleotides contemplated hereininclude single and double stranded DNA, single and double stranded RNA,and hybrid molecules having mixtures of single and double stranded DNAand RNA. Examples of nucleic acid, e.g., polynucleotides contemplatedherein include any types of RNA, e.g., mRNA, siRNA, miRNA, and guide RNAand any types of DNA, genomic DNA, plasmid DNA, and minicircle DNA, andany fragments thereof. The term “duplex” in the context ofpolynucleotides refers, in the usual and customary sense, to doublestrandedness. Nucleic acids can be linear or branched. For example,nucleic acids can be a linear chain of nucleotides or the nucleic acidscan be branched, e.g., such that the nucleic acids comprise one or morearms or branches of nucleotides. Optionally, the branched nucleic acidsare repetitively branched to form higher ordered structures such asdendrimers and the like. A “nucleic acid moiety” as used herein is amonovalent form of a nucleic acid. In embodiments, the nucleic acidmoiety is attached to the 3′ or 5′ position of a nucleotide ornucleoside.

Nucleic acids, including e.g., nucleic acids with a phosphorothioatebackbone, can include one or more reactive moieties. As used herein, theterm reactive moiety includes any group capable of reacting with anothermolecule, e.g., a nucleic acid or polypeptide through covalent,non-covalent or other interactions. By way of example, the nucleic acidcan include an amino acid reactive moiety that reacts with an amino acidon a protein or polypeptide through a covalent, non-covalent or otherinteraction.

As used herein, the term “template polynucleotide” refers to anypolynucleotide molecule that may be bound by a polymerase and utilizedas a template for nucleic acid synthesis. A template polynucleotide maybe a target polynucleotide. In general, the term “target polynucleotide”refers to a nucleic acid molecule or polynucleotide in a startingpopulation of nucleic acid molecules having a target sequence whosepresence, amount, and/or nucleotide sequence, or changes in one or moreof these, are desired to be determined. In general, the term “targetsequence” refers to a nucleic acid sequence on a single strand ofnucleic acid. The target sequence may be a portion of a gene, aregulatory sequence, genomic DNA, cDNA, RNA including mRNA, miRNA, rRNA,or others. The target sequence may be a target sequence from a sample ora secondary target such as a product of an amplification reaction. Atarget polynucleotide is not necessarily any single molecule orsequence. For example, a target polynucleotide may be any one of aplurality of target polynucleotides in a reaction, or allpolynucleotides in a given reaction, depending on the reactionconditions. For example, in a nucleic acid amplification reaction withrandom primers, all polynucleotides in a reaction may be amplified. As afurther example, a collection of targets may be simultaneously assayedusing polynucleotide primers directed to a plurality of targets in asingle reaction. As yet another example, all or a subset ofpolynucleotides in a sample may be modified by the addition of aprimer-binding sequence (such as by the ligation of adapters containingthe primer binding sequence), rendering each modified polynucleotide atarget polynucleotide in a reaction with the corresponding primerpolynucleotide(s). In the context of selective sequencing, “targetpolynucleotide(s)” refers to the subset of polynucleotide(s) to besequenced from within a starting population of polynucleotides.

“Nucleotide,” as used herein, refers to a nucleoside-5′-phosphate (e.g.,polyphosphate) compound, or a structural analog thereof, which can beincorporated (e.g., partially incorporated as anucleoside-5′-monophosphate or derivative thereof) by a nucleic acidpolymerase to extend a growing nucleic acid chain (such as a primer).Nucleotides may comprise bases such as adenine (A), cytosine (C),guanine (G), thymine (T), uracil (U), or analogues thereof, and maycomprise 1, 2, 3, 4, 5, 6, 7, 8, or more phosphates in the phosphategroup. Nucleotides may be modified at one or more of the base, sugar, orphosphate group. A nucleotide may have a label or tag attached (a“labeled nucleotide” or “tagged nucleotide”). In an embodiment, thenucleotide is a deoxyribonucleotide. In another embodiment, thenucleotide is a ribonucleotide. In embodiments, nucleotides comprise 3phosphate groups (e.g., a triphosphate group).

The terms also encompass nucleic acids containing known nucleotideanalogs or modified backbone residues or linkages, which are synthetic,naturally occurring, and non-naturally occurring, which have similarbinding properties as the reference nucleic acid, and which aremetabolized in a manner similar to the reference nucleotides. Examplesof such analogs include, without limitation, phosphodiester derivativesincluding, e.g., phosphoramidate, phosphorodiamidate, phosphorothioate(also known as phosphorothioate having double bonded sulfur replacingoxygen in the phosphate), phosphorodithioate, phosphonocarboxylic acids,phosphonocarboxylates, phosphonoacetic acid, phosphonoformic acid,methyl phosphonate, boron phosphonate, or O-methylphosphoroamiditelinkages (see, Eckstein, Oligonucleotides and Analogues: A PracticalApproach, Oxford University Press) as well as modifications to thenucleotide bases such as in 5-methyl cytidine or pseudouridine; andpeptide nucleic acid backbones and linkages. Other analog nucleic acidsinclude those with positive backbones; non-ionic backbones, modifiedsugars, and non-ribose backbones (e.g., phosphorodiamidate morpholinooligos or locked nucleic acids (LNA) as known in the art), includingthose described in U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters6 and 7, ASC Symposium Series 580, Carbohydrate Modifications inAntisense Research, Sanghui & Cook, eds. Nucleic acids containing one ormore carbocyclic sugars are also included within one definition ofnucleic acids. Modifications of the ribose-phosphate backbone may bedone for a variety of reasons, e.g., to increase the stability andhalf-life of such molecules in physiological environments or as probeson a biochip. Mixtures of naturally occurring nucleic acids and analogscan be made; alternatively, mixtures of different nucleic acid analogs,and mixtures of naturally occurring nucleic acids and analogs may bemade. In embodiments, the internucleotide linkages in DNA arephosphodiester, phosphodiester derivatives, or a combination of both.

In embodiments, “nucleotide analogue,” “nucleotide analog,” or“nucleotide derivative” shall mean an analogue of adenine (A), cytosine(C), guanine (G), thymine (T), or uracil (U) (that is, an analogue orderivative of a nucleotide comprising the base A, G, C, T or U),comprising a phosphate group, which may be recognized by DNA or RNApolymerase (whichever is applicable) and may be incorporated into astrand of DNA or RNA (whichever is appropriate). Examples of nucleotideanalogues include, without limitation, 7-deaza-adenine, 7-deaza-guanine,the analogues of deoxynucleotides shown herein, analogues in which alabel is attached through a cleavable linker to the 5-position ofcytosine or thymine or to the 7-position of deaza-adenine ordeaza-guanine, and analogues in which a small chemical moiety is used tocap the —OH group at the 3′-position of deoxyribose. Nucleotideanalogues and DNA polymerase-based DNA sequencing are also described inU.S. Pat. No. 6,664,079, which is incorporated herein by reference inits entirety for all purposes.

A “nucleoside” is structurally similar to a nucleotide, but is missingthe phosphate moieties. An example of a nucleoside analogue would be onein which the label is linked to the base and there is no phosphate groupattached to the sugar molecule. “Nucleoside,” as used herein, refers toa glycosyl compound consisting of a nucleobase and a 5-membered ringsugar (e.g., either ribose or deoxyribose). Nucleosides may comprisebases such as adenine (A), cytosine (C), guanine (G), thymine (T),uracil (U), or analogues thereof. Nucleosides may be modified at thebase and/or and the sugar. In an embodiment, the nucleoside is adeoxyribonucleoside. In another embodiment, the nucleoside is aribonucleoside.

A particular nucleic acid sequence also encompasses “splice variants.”Similarly, a particular protein encoded by a nucleic acid encompassesany protein encoded by a splice variant of that nucleic acid. “Splicevariants,” as the name suggests, are products of alternative splicing ofa gene. After transcription, an initial nucleic acid transcript may bespliced such that different (alternate) nucleic acid splice productsencode different polypeptides. Mechanisms for the production of splicevariants vary, but include alternate splicing of exons. Alternatepolypeptides derived from the same nucleic acid by read-throughtranscription are also encompassed by this definition. Any products of asplicing reaction, including recombinant forms of the splice products,are included in this definition. An example of potassium channel splicevariants is discussed in Leicher, et al., J. Biol. Chem.273(52):35095-35101 (1998).

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are near each other, and, inthe case of a secretory leader, contiguous and in reading phase.However, enhancers do not have to be contiguous. Linking is accomplishedby ligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

The terms “bioconjugate group,” “bioconjugate reactive moiety,” and“bioconjugate reactive group” refer to a chemical moiety whichparticipates in a reaction to form a bioconjugate linker (e.g., covalentlinker). Non-limiting examples of bioconjugate groups include —NH₂,—COOH, —COOCH₃, —N-hydroxysuccinimide, -maleimide,

In embodiments, the bioconjugate reactive group may be protected (e.g.,with a protecting group). In embodiments, the bioconjugate reactivemoiety is

or —NH₂. Additional examples of bioconjugate reactive groups and theresulting bioconjugate reactive linkers may be found in the BioconjugateTable below:

Bioconjugate reactive group 1 Bioconjugate reactive group 2 (e.g.,electrophilic bioconjugate (e.g., nucleophilic bioconjugate ResultingBioconjugate reactive moiety) reactive moiety) reactive linker activatedesters amines/anilines carboxamides acrylamides thiols thioethers acylazides amines/anilines carboxamides acyl halides amines/anilinescarboxamides acyl halides alcohols/phenols esters acyl nitrilesalcohols/phenols esters acyl nitriles amines/anilines carboxamidesaldehydes amines/anilines imines aldehydes or ketones hydrazineshydrazones aldehydes or ketones hydroxylamines oximes alkyl halidesamines/anilines alkyl amines alkyl halides carboxylic acids esters alkylhalides thiols thioethers alkyl halides alcohols/phenols ethers alkylsulfonates thiols thioethers alkyl sulfonates carboxylic acids estersalkyl sulfonates alcohols/phenols ethers anhydrides alcohols/phenolsesters anhydrides amines/anilines carboxamides aryl halides thiolsthiophenols aryl halides amines aryl amines aziridines thiols thioethersboronates glycols boronate esters carbodiimides carboxylic acidsN-acylureas or anhydrides diazoalkanes carboxylic acids esters epoxidesthiols thioethers haloacetamides thiols thioethers haloplatinate aminoplatinum complex haloplatinate heterocycle platinum complexhaloplatinate thiol platinum complex halotriazines amines/anilinesaminotriazines halotriazines alcohols/phenols triazinyl ethershalotriazines thiols triazinyl thioethers imido esters amines/anilinesamidines isocyanates amines/anilines ureas isocyanates alcohols/phenolsurethanes isothiocyanates amines/anilines thioureas maleimides thiolsthioethers phosphoramidites alcohols phosphite esters silyl halidesalcohols silyl ethers sulfonate esters amines/anilines alkyl aminessulfonate esters thiols thioethers sulfonate esters carboxylic acidsesters sulfonate esters alcohols ethers sulfonyl halides amines/anilinessulfonamides sulfonyl halides phenols/alcohols sulfonate esters

As used herein, the term “bioconjugate” or “bioconjugate linker” refersto the resulting association between atoms or molecules of bioconjugatereactive groups. The association can be direct or indirect. For example,a conjugate between a first bioconjugate reactive group (e.g., —NH₂,—COOH, —N-hydroxysuccinimide, or -maleimide) and a second bioconjugatereactive group (e.g., sulfhydryl, sulfur-containing amino acid, amine,amine sidechain containing amino acid, or carboxylate) provided hereincan be direct, e.g., by covalent bond or linker (e.g., a first linker ofsecond linker), or indirect, e.g., by non-covalent bond (e.g.,electrostatic interactions (e.g., ionic bond, hydrogen bond, halogenbond), van der Waals interactions (e.g., dipole-dipole, dipole-induceddipole, London dispersion), ring stacking (pi effects), hydrophobicinteractions and the like). In embodiments, bioconjugates orbioconjugate linkers are formed using bioconjugate chemistry (i.e., theassociation of two bioconjugate reactive groups) including, but are notlimited to nucleophilic substitutions (e.g., reactions of amines andalcohols with acyl halides, active esters), electrophilic substitutions(e.g., enamine reactions) and additions to carbon-carbon andcarbon-heteroatom multiple bonds (e.g., Michael reaction, Diels-Alderaddition). These and other useful reactions are discussed in, forexample, March, ADVANCED ORGANIC CHEMISTRY, 3rd Ed., John Wiley & Sons,New York, 1985; Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, SanDiego, 1996; and Feeney et al., MODIFICATION OF PROTEINS; Advances inChemistry Series, Vol. 198, American Chemical Society, Washington, D.C.,1982. In embodiments, the first bioconjugate reactive group (e.g.,maleimide moiety) is covalently attached to the second bioconjugatereactive group (e.g., a sulfhydryl). In embodiments, the firstbioconjugate reactive group (e.g., haloacetyl moiety) is covalentlyattached to the second bioconjugate reactive group (e.g., a sulfhydryl).In embodiments, the first bioconjugate reactive group (e.g., pyridylmoiety) is covalently attached to the second bioconjugate reactive group(e.g., a sulfhydryl). In embodiments, the first bioconjugate reactivegroup (e.g., —N-hydroxysuccinimide moiety) is covalently attached to thesecond bioconjugate reactive group (e.g., an amine). In embodiments, thefirst bioconjugate reactive group (e.g., maleimide moiety) is covalentlyattached to the second bioconjugate reactive group (e.g., a sulfhydryl).In embodiments, the first bioconjugate reactive group (e.g.,-sulfo-N-hydroxysuccinimide moiety) is covalently attached to the secondbioconjugate reactive group (e.g., an amine). In embodiments, the firstbioconjugate reactive group (e.g., maleimide moiety) is covalentlyattached to the second bioconjugate reactive group (e.g., a sulfhydryl).In embodiments, the first bioconjugate reactive group (e.g.,-sulfo-N-hydroxysuccinimide moiety) is covalently attached to the secondbioconjugate reactive group (e.g., an amine). In embodiments, the firstbioconjugate reactive group (e.g., —COOH) is covalently attached to thesecond bioconjugate reactive group (e.g.,

thereby forming a bioconjugate (e.g.,

In embodiments, the first bioconjugate reactive group (e.g., —NH₂) iscovalently attached to the second bioconjugate reactive group (e.g.,

thereby forming a bioconjugate (e.g.,

In embodiments, the first bioconjugate reactive group (e.g., a couplingreagent) is covalently attached to the second bioconjugate reactivegroup (e.g.,

thereby forming a bioconjugate (e.g.,

The bioconjugate reactive groups can be chosen such that they do notparticipate in, or interfere with, the chemical stability of theconjugate described herein. Alternatively, a reactive functional groupcan be protected from participating in the crosslinking reaction by thepresence of a protecting group. In embodiments, the bioconjugatecomprises a molecular entity derived from the reaction of an unsaturatedbond, such as a maleimide, and a sulfhydryl group.

Useful bioconjugate reactive groups used for bioconjugate chemistriesherein include, for example: (a) carboxyl groups and various derivativesthereof including, but not limited to, N-hydroxysuccinimide esters,N-hydroxybenztriazole esters, acid halides, acyl imidazoles, thioesters,p-nitrophenyl esters, alkyl, alkenyl, alkynyl and aromatic esters; (b)hydroxyl groups which can be converted to esters, ethers, aldehydes,etc.; (c) haloalkyl groups wherein the halide can be later displacedwith a nucleophilic group such as, for example, an amine, a carboxylateanion, thiol anion, carbanion, or an alkoxide ion, thereby resulting inthe covalent attachment of a new group at the site of the halogen atom;(d) dienophile groups which are capable of participating in Diels-Alderreactions such as, for example, maleimido or maleimide groups; (e)aldehyde or ketone groups such that subsequent derivatization ispossible via formation of carbonyl derivatives such as, for example,imines, hydrazones, semicarbazones or oximes, or via such mechanisms asGrignard addition or alkyllithium addition; (f) sulfonyl halide groupsfor subsequent reaction with amines, for example, to form sulfonamides;(g) thiol groups, which can be converted to disulfides, reacted withacyl halides, or bonded to metals such as gold, or react withmaleimides; (h) amine or sulfhydryl groups (e.g., present in cysteine),which can be, for example, acylated, alkylated or oxidized; (i) alkenes,which can undergo, for example, cycloadditions, acylation, Michaeladdition, etc.; (j) epoxides, which can react with, for example, aminesand hydroxyl compounds; (k) phosphoramidites and other standardfunctional groups useful in nucleic acid synthesis; (l) metal siliconoxide bonding; (m) metal bonding to reactive phosphorus groups (e.g.,phosphines) to form, for example, phosphate diester bonds; (n) azidescoupled to alkynes using copper catalyzed cycloaddition click chemistry;(o) biotin conjugate can react with avidin or streptavidin to form aavidin-biotin complex or streptavidin-biotin complex.

The term “monophosphate” is used in accordance with its ordinary meaningin the arts and refers to a moiety having the formula:

or ionized forms thereof. The term “polyphosphate” refers to at leasttwo phosphate groups, having the formula:

or ionized forms thereof, wherein np is an integer of 1 or greater. Inembodiments, np is an integer from 1 to 5. In embodiments, np is aninteger from 1 to 2. In embodiments, np is 2. The term “diphosphate” isused in accordance with its ordinary meaning in the arts and refers to amoiety having the formula:

or ionized forms thereof. The term “triphosphate” is used in accordancewith its ordinary meaning in the arts and refers to a moiety having theformula:

or ionized forms thereof. In embodiments, a polyphosphate is adiphosphate. In embodiments, a polyphosphate is a triphosphate.

The term “nucleobase” or “base” as used herein refers to a purine orpyrimidine compound, or a derivative thereof, that may be a constituentof nucleic acid (i.e., DNA or RNA, or a derivative thereof). Inembodiments, the nucleobase is a divalent purine or pyrimidine, orderivative thereof. In embodiments, the nucleobase is a monovalentpurine or pyrimidine, or derivative thereof. In embodiments, the base isa derivative of a naturally occurring DNA or RNA base (e.g., a baseanalogue). In embodiments the base is a hybridizing base. In embodimentsthe base hybridizes to a complementary base. In embodiments, the base iscapable of forming at least one hydrogen bond with a complementary base(e.g., adenine hydrogen bonds with thymine, adenine hydrogen bonds withuracil, guanine pairs with cytosine). Non-limiting examples of a baseincludes cytosine or a derivative thereof (e.g., cytosine analogue),guanine or a derivative thereof (e.g., guanine analogue), adenine or aderivative thereof (e.g., adenine analogue), thymine or a derivativethereof (e.g., thymine analogue), uracil or a derivative thereof (e.g.,uracil analogue), hypoxanthine or a derivative thereof (e.g.,hypoxanthine analogue), xanthine or a derivative thereof (e.g., xanthineanalogue), 7-methylguanine or a derivative thereof (e.g.,7-methylguanine analogue), deaza-adenine or a derivative thereof (e.g.,deaza-adenine analogue), deaza-guanine or a derivative thereof (e.g.,deaza-guanine), deaza-hypoxanthine or a derivative thereof,5,6-dihydrouracil or a derivative thereof (e.g., 5,6-dihydrouracilanalogue), 5-methylcytosine or a derivative thereof (e.g.,5-methylcytosine analogue), or 5-hydroxymethylcytosine or a derivativethereof (e.g., 5-hydroxymethylcytosine analogue) moieties. Inembodiments, the base is adenine, guanine, uracil, cytosine, thymine,hypoxanthine, xanthine, theobromine, caffeine, uric acid, or isoguanine,which may be optionally substituted or modified. In embodiments, thebase is adenine, guanine, hypoxanthine, xanthine, theobromine, caffeine,uric acid, or isoguanine, which may be optionally substituted ormodified.

As used herein, the term “complementary” or “substantiallycomplementary” refers to the hybridization, base pairing, or theformation of a duplex between nucleotides or nucleic acids. For example,complementarity exists between the two strands of a double-stranded DNAmolecule or between an oligonucleotide primer and a primer binding siteon a single-stranded nucleic acid when a nucleotide (e.g., RNA or DNA)or a sequence of nucleotides is capable of base pairing with arespective cognate nucleotide or cognate sequence of nucleotides. Asdescribed herein and commonly known in the art the complementary(matching) nucleotide of adenosine (A) is thymidine (T) and thecomplementary (matching) nucleotide of guanosine (G) is cytosine (C).Thus, a complement may include a sequence of nucleotides that base pairwith corresponding complementary nucleotides of a second nucleic acidsequence. The nucleotides of a complement may partially or completelymatch the nucleotides of the second nucleic acid sequence. Where thenucleotides of the complement completely match each nucleotide of thesecond nucleic acid sequence, the complement forms base pairs with eachnucleotide of the second nucleic acid sequence. Where the nucleotides ofthe complement partially match the nucleotides of the second nucleicacid sequence only some of the nucleotides of the complement form basepairs with nucleotides of the second nucleic acid sequence. Examples ofcomplementary sequences include coding and non-coding sequences, whereinthe non-coding sequence contains complementary nucleotides to the codingsequence and thus forms the complement of the coding sequence. A furtherexample of complementary sequences are sense and antisense sequences,wherein the sense sequence contains complementary nucleotides to theantisense sequence and thus forms the complement of the antisensesequence. “Duplex” means at least two oligonucleotides and/orpolynucleotides that are fully or partially complementary undergoWatson-Crick type base pairing among all or most of their nucleotides sothat a stable complex is formed.

As described herein, the complementarity of sequences may be partial, inwhich only some of the nucleic acids match according to base pairing, orcomplete, where all the nucleic acids match according to base pairing.Thus, two sequences that are complementary to each other, may have aspecified percentage of nucleotides that complement one another (e.g.,about 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or higher complementarity over a specifiedregion). In embodiments, two sequences are complementary when they arecompletely complementary, having 100% complementarity. In embodiments,sequences in a pair of complementary sequences form portions of a singlepolynucleotide with non-base-pairing nucleotides (e.g., as in a hairpinor loop structure, with or without an overhang) or portions of separatepolynucleotides. In embodiments, one or both sequences in a pair ofcomplementary sequences form portions of longer polynucleotides, whichmay or may not include additional regions of complementarity.

The term “non-covalent linker” is used in accordance with its ordinarymeaning and refers to a divalent moiety which includes at least twomolecules that are not covalently linked to each other but are capableof interacting with each other via a non-covalent bond (e.g.,electrostatic interactions (e.g., ionic bond, hydrogen bond, halogenbond) or van der Waals interactions (e.g., dipole-dipole, dipole-induceddipole, London dispersion). In embodiments, the non-covalent linker isthe result of two molecules that are not covalently linked to each otherthat interact with each other via a non-covalent bond.

The term “anchor moiety” as used herein refers to a chemical moietycapable of interacting (e.g., covalently or non-covalently) with asecond, optionally different, chemical moiety (e.g., complementaryanchor moiety binder). In embodiments, the anchor moiety is abioconjugate reactive group capable of interacting (e.g., covalently)with a complementary bioconjugate reactive group (e.g., complementaryanchor moiety reactive group, complementary anchor moiety binder). Inembodiments, an anchor moiety is a click chemistry reactant moiety. Inembodiments, the anchor moiety (an “affinity anchor moiety”) is capableof non-covalently interacting with a second chemical moiety (e.g.,complementary affinity anchor moiety binder). Non-limiting examples ofan anchor moiety include biotin, azide, trans-cyclooctene (TCO)(Blackman, M. L., et al., J. Am. Chem. Soc., 2008, 130, 13518-13519;Debets, M. F., et al. Org. Biomol. Chem., 2013, 11, 6439-6455) andphenyl boric acid (PBA) (Bergseid M., et al., BioTechniques, 2000, 29,1126-1133). In embodiments, an affinity anchor moiety (e.g., biotinmoiety) interacts non-covalently with a complementary affinity anchormoiety binder (e.g., streptavidin moiety). In embodiments, an anchormoiety (e.g., azide moiety, trans-cyclooctene (TCO) moiety, phenyl boricacid (PBA) moiety) covalently binds a complementary anchor moiety binder(e.g., dibenzocyclooctyne (DBCO) moiety (Jewett J. C. and Bertozzi C. R.J. Am. Chem. Soc., 2010, 132, 3688-3690), tetrazine (TZ) moiety,salicylhydroxamic acid (SHA) moiety).

The term “cleavable linker” or “cleavable moiety” as used herein refersto a divalent or monovalent, respectively, moiety which is capable ofbeing separated (e.g., detached, split, disconnected, hydrolyzed, astable bond within the moiety is broken) into distinct entities. Inembodiments, a cleavable linker is cleavable (e.g., specificallycleavable) in response to external stimuli (e.g., enzymes,nucleophilic/basic reagents, reducing agents, photo-irradiation,electrophilic/acidic reagents, organometallic and metal reagents, oroxidizing reagents). In embodiments, a cleavable linker is aself-immolative linker, a trivalent linker, or a linker capable ofdendritic amplication of signal, or a self-immolative dendrimercontaining linker (e.g., all as described in US 2007/0009980, US2006/0003383, and US 2009/0047699, which are incorporated by referencein their entirety for any purpose). A chemically cleavable linker refersto a linker which is capable of being split in response to the presenceof a chemical (e.g., acid, base, oxidizing agent, reducing agent, Pd(0),tris-(2-carboxyethyl)phosphine, dilute nitrous acid, fluoride,tris(3-hydroxypropyl)phosphine), sodium dithionite (Na₂S₂O₄), hydrazine(N₂H₄)). A chemically cleavable linker is non-enzymatically cleavable.In embodiments, the cleavable linker is cleaved by contacting thecleavable linker with a cleaving agent. In embodiments, the cleavingagent is sodium dithionite (Na₂S₂O₄), weak acid, hydrazine (N₂H₄),Pd(0), or light-irradiation (e.g., ultraviolet radiation). Inembodiments, cleaving includes removing. A “cleavable site” or “scissilelinkage” in the context of a polynucleotide is a site which allowscontrolled cleavage of the polynucleotide strand (e.g., the linker, theprimer, or the polynucleotide) by chemical, enzymatic, or photochemicalmeans known in the art and described herein. A scissile site may referto the linkage of a nucleotide between two other nucleotides in anucleotide strand (i.e., an internucleosidic linkage). In embodiments,the scissile linkage can be located at any position within the one ormore nucleic acid molecules, including at or near a terminal end (e.g.,the 3′ end of an oligonucleotide) or in an interior portion of the oneor more nucleic acid molecules. In embodiments, conditions suitable forseparating a scissile linkage include a modulating the pH and/or thetemperature. In embodiments, a scissile site can include at least oneacid-labile linkage. For example, an acid-labile linkage may include aphosphoramidate linkage. In embodiments, a phosphoramidate linkage canbe hydrolysable under acidic conditions, including mild acidicconditions such as trifluoroacetic acid and a suitable temperature(e.g., 30° C.), or other conditions known in the art, for exampleMatthias Mag, et al Tetrahedron Letters, Volume 33, Issue 48, 1992,7319-7322. In embodiments, the scissile site can include at least onephotolabile internucleosidic linkage (e.g., o-nitrobenzyl linkages, asdescribed in Walker et al, J. Am. Chem. Soc. 1988, 110, 21, 7170-7177),such as o-nitrobenzyloxymethyl or p-nitrobenzyloxymethyl group(s). Inembodiments, the scissile site includes at least one uracil nucleobase.In embodiments, a uracil nucleobase can be cleaved with a uracil DNAglycosylase (UDG) or Formamidopyrimidine DNA Glycosylase Fpg. Inembodiments, the scissile linkage site includes a sequence-specificnicking site having a nucleotide sequence that is recognized and nickedby a nicking endonuclease enzyme or a uracil DNA glycosylase. The term“self-immolative”referring to a linker is used in accordance with itswell understood meaning in Chemistry and Biology as used in US2007/0009980, US 2006/0003383, and US 2009/0047699, which areincorporated by reference in their entirety for any purpose. Inembodiments “self-immolative” referring to a linker refers to a linkerthat is capable of additional cleavage following initial cleavage by anexternal stimuli. The term dendrimer is used in accordance with its wellunderstood meaning in Chemistry. In embodiments, the term“self-immolative dendrimer” is used as described in US 2007/0009980, US2006/0003383, and US 2009/0047699, which are incorporated by referencein their entirety for any purpose and in embodiments refers to adendrimer that is capable of releasing all of its tail units through aself-immolative fragmentation following initial cleavage by an externalstimulus.

A “photocleavable linker” (e.g., including or consisting of ano-nitrobenzyl group) refers to a linker which is capable of being splitin response to photo-irradiation (e.g., ultraviolet radiation). Anacid-cleavable linker refers to a linker which is capable of being splitin response to a change in the pH (e.g., increased acidity). Abase-cleavable linker refers to a linker which is capable of being splitin response to a change in the pH (e.g., decreased acidity). Anoxidant-cleavable linker refers to a linker which is capable of beingsplit in response to the presence of an oxidizing agent. Areductant-cleavable linker refers to a linker which is capable of beingsplit in response to the presence of a reducing agent (e.g.,tris(3-hydroxypropyl)phosphine). In embodiments, the cleavable linker isa dialkylketal linker (Binaulda S., et al., Chem. Commun., 2013, 49,2082-2102; Shenoi R. A., et al., J. Am. Chem. Soc., 2012, 134,14945-14957), an azo linker (Rathod, K. M., et al., Chem. Sci. Tran.,2013, 2, 25-28; Leriche G., et al., Eur. J. Org. Chem., 2010, 23,4360-64), an allyl linker, a cyanoethyl linker, a1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl linker, or anitrobenzyl linker.

The term “orthogonally cleavable linker” or “orthogonal cleavablelinker” as used herein refer to a cleavable linker that is cleaved by afirst cleaving agent (e.g., enzyme, nucleophilic/basic reagent, reducingagent, photo-irradiation, electrophilic/acidic reagent, organometallicand metal reagent, oxidizing reagent) in a mixture of two or moredifferent cleaving agents and is not cleaved by any other differentcleaving agent in the mixture of two or more cleaving agents. Forexample, two different cleavable linkers are both orthogonal cleavablelinkers when a mixture of the two different cleavable linkers arereacted with two different cleaving agents and each cleavable linker iscleaved by only one of the cleaving agents and not the other cleavingagent and the agent that cleaves each cleavable linker is different. Inembodiments, an orthogonally is a cleavable linker that followingcleavage the two separated entities (e.g., fluorescent dye, bioconjugatereactive group) do not further react and form a new orthogonallycleavable linker.

The term “orthogonal detectable label” or “orthogonal detectable moiety”as used herein refer to a detectable label (e.g., fluorescent dye ordetectable dye) that is capable of being detected and identified (e.g.,by use of a detection means (e.g., emission wavelength, physicalcharacteristic measurement)) in a mixture or a panel (collection ofseparate samples) of two or more different detectable labels. Forexample, two different detectable labels that are fluorescent dyes areboth orthogonal detectable labels when a panel of the two differentfluorescent dyes is subjected to a wavelength of light that is absorbedby one fluorescent dye but not the other and results in emission oflight from the fluorescent dye that absorbed the light but not the otherfluorescent dye. Orthogonal detectable labels may be separatelyidentified by different absorbance or emission intensities of theorthogonal detectable labels compared to each other and not only be theabsolute presence of absence of a signal. An example of a set of fourorthogonal detectable labels is the set of Rox-Labeled Tetrazine,Alexa488-Labeled SHA, Cy5-Labeled Streptavidin, and R6G-LabeledDibenzocyclooctyne.

The term “polymerase-compatible cleavable moiety” or “reversibleterminator” as used herein refers to a cleavable moiety which does notinterfere with a function of a polymerase (e.g., DNA polymerase,modified DNA polymerase, in incorporating the nucleotide, to which thepolymerase-compatible cleavable moiety is attached, to the 3′ end of thenewly formed nucleotide strand). Methods for determining the function ofa polymerase contemplated herein are described in B. Rosenblum et al.(Nucleic Acids Res. 1997 Nov. 15; 25(22): 4500-4504); and Z. Zhu et al.(Nucleic Acids Res. 1994 Aug. 25; 22(16): 3418-3422), which areincorporated by reference herein in their entirety for all purposes. Inembodiments the polymerase-compatible cleavable moiety does not decreasethe function of a polymerase relative to the absence of thepolymerase-compatible cleavable moiety. In embodiments, thepolymerase-compatible cleavable moiety does not negatively affect DNApolymerase recognition. In embodiments, the polymerase-compatiblecleavable moiety does not negatively affect (e.g., limit) the readlength of the DNA polymerase. Additional examples of apolymerase-compatible cleavable moiety may be found in U.S. Pat. No.6,664,079, Ju J. et al. (2006) Proc Natl Acad Sci USA103(52):19635-19640; Ruparel H. et al. (2005) Proc Natl Acad Sci USA102(17):5932-5937; Wu J. et al. (2007) Proc Natl Acad Sci USA104(104):16462-16467; Guo J. et al. (2008) Proc Natl Acad Sci USA105(27): 9145-9150 Bentley D. R. et al. (2008) Nature 456(7218):53-59;or Hutter D. et al. (2010) Nucleosides Nucleotides & Nucleic Acids29:879-895, which are incorporated herein by reference in their entiretyfor all purposes. In embodiments, a polymerase-compatible cleavablemoiety includes an azido moiety or a dithiol linking moiety. Inembodiments, the polymerase-compatible cleavable moiety is —NH₂, —CN,—CH₃, C₂-C₆ allyl (e.g., —CH₂—CH═CH₂), methoxyalkyl (e.g., —CH₂—O—CH₃),or —CH₂N₃. In embodiments, the polymerase-compatible cleavable moietycomprises a disulfide moiety. In embodiments, a polymerase-compatiblecleavable moiety is a cleavable moiety on a nucleotide, nucleobase,nucleoside, or nucleic acid that does not interfere with a function of apolymerase (e.g., DNA polymerase, modified DNA polymerase).

The term “allyl” as described herein refers to an unsubstitutedmethylene attached to a vinyl group (i.e., —CH═CH₂), having the formula

An “allyl linker” refers to a divalent unsubstituted methylene attachedto a vinyl group, having the formula

The term “polymer” refers to a molecule including repeating subunits(e.g., polymerized monomers). For example, polymeric molecules may bebased upon polyethylene glycol (PEG), tetraethylene glycol (TEG),polyvinylpyrrolidone (PVP), poly(xylene), or poly(p-xylylene). The term“polymerizable monomer” is used in accordance with its meaning in theart of polymer chemistry and refers to a compound that may covalentlybind chemically to other monomer molecules (such as other polymerizablemonomers that are the same or different) to form a polymer.

The term “polymerase-compatible moiety” as used herein refers a moietywhich does not interfere with the function of a polymerase (e.g., DNApolymerase, modified DNA polymerase) in incorporating the nucleotide towhich the polymerase-compatible moiety is attached to the 3′ end of thenewly formed nucleotide strand. The polymerase-compatible moiety does,however, interfere with the polymerase function by preventing theaddition of another nucleotide to the 3′ oxygen of the nucleotide towhich the polymerase-compatible moiety is attached. Methods fordetermining the function of a polymerase contemplated herein aredescribed in B. Rosenblum et al. (Nucleic Acids Res. 1997 Nov. 15;25(22): 4500-4504); and Z. Zhu et al. (Nucleic Acids Res. 1994 Aug. 25;22(16): 3418-3422), which are incorporated by reference herein in theirentirety for all purposes. In embodiments the polymerase-compatiblemoiety does not decrease the function of a polymerase relative to theabsence of the polymerase-compatible moiety. In embodiments, thepolymerase-compatible moiety does not negatively affect DNA polymeraserecognition. In embodiments, the polymerase-compatible moiety does notnegatively affect (e.g., limit) the read length of the DNA polymerase.Additional examples of a polymerase-compatible moiety may be found inU.S. Pat. No. 6,664,079, Ju J. et al. (2006) Proc Natl Acad Sci USA103(52):19635-19640; Ruparel H. et al. (2005) Proc Natl Acad Sci USA102(17):5932-5937; Wu J. et al. (2007) Proc Natl Acad Sci USA104(104):16462-16467; Guo J. et al. (2008) Proc Natl Acad Sci USA105(27): 9145-9150 Bentley D. R. et al. (2008) Nature 456(7218):53-59;or Hutter D. et al. (2010) Nucleosides Nucleotides & Nucleic Acids29:879-895, which are incorporated herein by reference in their entiretyfor all purposes. In embodiments, a polymerase-compatible moietyincludes hydrogen, —N₃, —CN, or halogen. In embodiments, apolymerase-compatible moiety is a moiety on a nucleotide, nucleobase,nucleoside, or nucleic acid that does not interfere with the function ofa polymerase (e.g., DNA polymerase, modified DNA polymerase).

As used herein, the term “DNA polymerase” and “nucleic acid polymerase”are used in accordance with their plain ordinary meanings and refer toenzymes capable of synthesizing nucleic acid molecules from nucleotides(e.g., deoxyribonucleotides). Typically, a DNA polymerase addsnucleotides to the 3′-end of a DNA strand, one nucleotide at a time. Inembodiments, the DNA polymerase is a Pol I DNA polymerase, Pol II DNApolymerase, Pol III DNA polymerase, Pol IV DNA polymerase, Pol V DNApolymerase, Pol R DNA polymerase, Pol μ DNA polymerase, Pol λ DNApolymerase, Pol σ DNA polymerase, Pol α DNA polymerase, Pol δ DNApolymerase, Pol ε DNA polymerase, Pol η DNA polymerase, Pol ι DNApolymerase, Pol κ DNA polymerase, Pol ζ DNA polymerase, Pol γ DNApolymerase, Pol θ DNA polymerase, Pol υ DNA polymerase, or athermophilic nucleic acid polymerase (e.g. Therminator γ, 9° Npolymerase (exo-), Therminator II, Therminator III, or Therminator IX).In embodiments, the DNA polymerase is a modified archaeal DNApolymerase. In embodiments, the polymerase is a reverse transcriptase.In embodiments, the polymerase is a mutant P. abyssi polymerase (e.g.,such as a mutant P. abyssi polymerase described in WO 2018/148723 or WO2020/056044).

As used herein, the term “thermophilic nucleic acid polymerase” refersto a family of DNA polymerases (e.g., 9° N™) and mutants thereof derivedfrom the DNA polymerase originally isolated from the hyperthermophilicarchaea, Thermococcus sp. 9 degrees N-7, found in hydrothermal vents atthat latitude (East Pacific Rise) (Southworth M W, et al. PNAS. 1996;93(11):5281-5285). A thermophilic nucleic acid polymerase is a member ofthe family B DNA polymerases. Site-directed mutagenesis of the 3′-5′ exomotif I (Asp-Ile-Glu or DIE) to AIA, AIE, EIE, EID or DIA yieldedpolymerase with no detectable 3′ exonuclease activity. Mutation toAsp-Ile-Asp (DID) resulted in reduction of 3′-5′ exonuclease specificactivity to <1% of wild type, while maintaining other properties of thepolymerase including its high strand displacement activity. The sequenceAIA (D141A, E143A) was chosen for reducing exonuclease. Subsequentmutagenesis of key amino acids results in an increased ability of theenzyme to incorporate dideoxynucleotides, ribonucleotides andacyclonucleotides (e.g., Therminator II enzyme from New England Biolabswith D141A/E143A/Y409V/A485L mutations); 3′-amino-dNTPs, 3′-azido-dNTPsand other 3′-modified nucleotides (e.g., NEB Therminator III DNAPolymerase with D141A/E143A/L408S/Y409A/P410V mutations, NEB TherminatorIX DNA polymerase), or 7-phosphate labeled nucleotides (e.g.,Therminator γ:D141A/E143A/W355A/L408W/R460A/Q461S/K464E/D480V/R484W/A485L). Typically,these enzymes do not have 5′-3′ exonuclease activity. Additionalinformation about thermophilic nucleic acid polymerases may be found in(Southworth M W, et al. PNAS. 1996; 93(11):5281-5285; Bergen K, et al.Chem Bio Chem. 2013; 14(9):1058-1062; Kumar S, et al. ScientificReports. 2012; 2:684; Fuller C W, et al. 2016; 113(19):5233-5238; Guo J,et al. Proceedings of the National Academy of Sciences of the UnitedStates of America. 2008; 105(27):9145-9150), which are incorporatedherein in their entirety for all purposes.

As used herein, the term “exonuclease activity” is used in accordancewith its ordinary meaning in the art, and refers to the removal of anucleotide from a nucleic acid by a DNA polymerase. For example, duringpolymerization, nucleotides are added to the 3′ end of the primerstrand. Occasionally a DNA polymerase incorporates an incorrectnucleotide to the 3′-OH terminus of the primer strand, wherein theincorrect nucleotide cannot form a hydrogen bond to the correspondingbase in the template strand. Such a nucleotide, added in error, isremoved from the primer as a result of the 3′ to 5′ exonuclease activityof the DNA polymerase. In embodiments, exonuclease activity may bereferred to as “proofreading.” When referring to 3′-5′ exonucleaseactivity, it is understood that the DNA polymerase facilitates ahydrolyzing reaction that breaks phosphodiester bonds at the 3′ end of apolynucleotide chain to excise the nucleotide. In embodiments, 3′-5′exonuclease activity refers to the successive removal of nucleotides insingle-stranded DNA in a 3′→5′ direction, releasing deoxyribonucleoside5′-monophosphates one after another. Methods for quantifying exonucleaseactivity are known in the art, see for example Southworth et al, PNASVol 93, 8281-8285 (1996).

As used herein, the terms “polynucleotide primer” and “primer” refers toany polynucleotide molecule that may hybridize to a polynucleotidetemplate, be bound by a polymerase, and be extended in atemplate-directed process for nucleic acid synthesis. The primer may bea separate polynucleotide from the polynucleotide template, or both maybe portions of the same polynucleotide (e.g., as in a hairpin structurehaving a 3′ end that is extended along another portion of thepolynucleotide to extend a double-stranded portion of the hairpin).Primers (e.g., forward or reverse primers) may be attached to a solidsupport. A primer can be of any length depending on the particulartechnique it will be used for. For example, PCR primers are generallybetween 10 and 40 nucleotides in length. The length and complexity ofthe nucleic acid fixed onto the nucleic acid template may vary. In someembodiments, a primer has a length of 200 nucleotides or less. Incertain embodiments, a primer has a length of 10 to 150 nucleotides, 15to 150 nucleotides, 5 to 100 nucleotides, 5 to 50 nucleotides or 10 to50 nucleotides. One of skill can adjust these factors to provide optimumhybridization and signal production for a given hybridization procedure.The primer permits the addition of a nucleotide residue thereto, oroligonucleotide or polynucleotide synthesis therefrom, under suitableconditions. In an embodiment the primer is a DNA primer, i.e., a primerconsisting of, or largely consisting of, deoxyribonucleotide residues.The primers are designed to have a sequence that is the complement of aregion of template/target DNA to which the primer hybridizes. Theaddition of a nucleotide residue to the 3′ end of a primer by formationof a phosphodiester bond results in a DNA extension product. Theaddition of a nucleotide residue to the 3′ end of the DNA extensionproduct by formation of a phosphodiester bond results in a further DNAextension product. In another embodiment the primer is an RNA primer. Inembodiments, a primer is hybridized to a target polynucleotide. A“primer” is complementary to a polynucleotide template, and complexes byhydrogen bonding or hybridization with the template to give aprimer/template complex for initiation of synthesis by a polymerase,which is extended by the addition of covalently bonded bases linked atits 3′ end complementary to the template in the process of DNAsynthesis.

“Polymerase,” as used herein, refers to any natural or non-naturallyoccurring enzyme or other catalyst that is capable of catalyzing apolymerization reaction, such as the polymerization of nucleotidemonomers to form a nucleic acid polymer. Exemplary types of polymerasesthat may be used in the compositions and methods of the presentdisclosure include the nucleic acid polymerases such as DNA polymerase,DNA- or RNA-dependent RNA polymerase, and reverse transcriptase. In somecases, the DNA polymerase is 9° N polymerase or a variant thereof, E.Coli DNA polymerase I, Bacteriophage T4 DNA polymerase, Sequenase, TaqDNA polymerase, DNA polymerase from Bacillus stearothermophilus, Bst 2.0DNA polymerase, 9° N polymerase, 9° N polymerase (exo-)A485L/Y409V,Phi29 DNA Polymerase (φ29 DNA Polymerase), T7 DNA polymerase, DNApolymerase II, DNA polymerase III holoenzyme, DNA polymerase IV, DNApolymerase V, VentR DNA polymerase, Therminator™ II DNA Polymerase,Therminator™ III DNA Polymerase, or or Therminator™ IX DNA Polymerase.In embodiments, the polymerase is a protein polymerase.

The phrase “stringent hybridization conditions” refers to conditionsunder which a primer will hybridize to its target subsequence, typicallyin a complex mixture of nucleic acids, but to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength pH. The T_(m) is the temperature (under definedionic strength, pH, and nucleic concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at T_(m),50% of the probes are occupied at equilibrium). Stringent conditions mayalso be achieved with the addition of destabilizing agents such asformamide. For selective or specific hybridization, a positive signal isat least two times background, preferably 10 times backgroundhybridization. Exemplary stringent hybridization conditions can be asfollowing: 50% formamide, 5×SSC, and 1% SDS, incubating at 42° C., or,5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDSat 65° C.

“Solid substrate” shall mean any suitable medium present in the solidphase to which a nucleic acid or an agent may be affixed. Non-limitingexamples include chips, beads and columns. The solid substrate can benon-porous or porous. Exemplary solid substrates include, but are notlimited to, glass and modified or functionalized glass, plastics(including acrylics, polystyrene and copolymers of styrene and othermaterials, polypropylene, polyethylene, polybutylene, polyurethanes,Teflon™, cyclic olefins, polyimides, etc.), nylon, ceramics, resins,Zeonor, silica or silica-based materials including silicon and modifiedsilicon, carbon, metals, inorganic glasses, optical fiber bundles, andpolymers. In embodiments, the solid substrate for have at least onesurface located within a flow cell. The solid substrate, or regionsthereof, can be substantially flat. The solid substrate can have surfacefeatures such as wells, pits, channels, ridges, raised regions, pegs,posts or the like. The term solid substrate is encompassing of asubstrate (e.g., a flow cell) having a surface comprising a polymercoating covalently attached thereto. In embodiments, the solid substrateis a flow cell. The term “flowcell” or “flow cell” as used herein refersto a chamber including a solid surface across which one or more fluidreagents can be flowed. Examples of flowcells and related fluidicsystems and detection platforms that can be readily used in the methodsof the present disclosure are described, for example, in Bentley et al.,Nature 456:53-59 (2008).

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. A positive hybridization is at least twicebackground. Those of ordinary skill will readily recognize thatalternative hybridization and wash conditions can be utilized to provideconditions of similar stringency. Additional guidelines for determininghybridization parameters are provided in numerous references, e.g.,Current Protocols in Molecular Biology, ed. Ausubel, et al., supra.

The term “thio-trigger moiety” refers to a substituent having theformula

wherein X is —O—, —NH—, or —S—; R¹⁰⁰ is —SO₃H, —SR¹⁰² or —CN; and R¹⁰²and R^(102a) are independently hydrogen, halogen, —CCl₃, —CBr₃, —CF₃,—CI₃, —CHCl₂, —CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN,—OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCCl₃, —OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl. Inembodiments, the thio-trigger moiety has the formula:

wherein X is —O—, —NH—, or —S—; R¹⁰⁰ is —SR¹⁰² or —CN; and R¹⁰² andR^(102a) are independently hydrogen, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃,—CHCl₂, —CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl. Inembodiments, the thio-trigger moiety has the formula:

wherein X is —O—, and R¹⁰⁰ and R^(102a) are as described herein. Inembodiments, the thio-trigger moiety has the formula:

wherein X is —NH—, and R¹⁰⁰ and R^(102a) are as described herein.

A “thio-trigger containing linker” refers to a covalent linker thatincludes a thio-trigger moiety. When a reducing agent (e.g.,dithiothreitol, THPP, or TCEP) contacts a thio-trigger containinglinker, the heteroatom represented by the symbol X (e.g., oxygen) of thethio-trigger moiety is reduced, and breaks the linker apart, accordingto the example mechanism:

R², R³, R⁴, R¹⁰⁰, R^(102a), L¹⁰¹, L¹⁰³, L¹⁰⁴, and L¹⁰⁵ are as describedherein, including in embodiments.

Where a range of values is provided herein, it is understood that eachintervening value, to the tenth of the unit (if appropriate) of thelower limit unless the context clearly dictates otherwise, between theupper and lower limit of that range, and any other stated or interveningvalue in that stated range, is encompassed within the invention. Theupper and lower limits of these smaller ranges may independently beincluded in the smaller ranges, and are also encompassed within theinvention, subject to any specifically excluded limit in the statedrange. Where the stated range includes one or both of the limits, rangesexcluding either or both of those included limits are also included inthe invention.

While various embodiments of the invention are shown and describedherein, it will be understood by those skilled in the art that suchembodiments are provided by way of example only. Numerous variations,changes, and substitutes may occur to those skilled in the art withoutdeparting from the invention. It should be understood that variousalternatives to the embodiments of the invention described herein may beemployed.

As used herein, and unless stated otherwise, each of the following termsshall be used in accordance with their plain and ordinary meaning, forexample: A indicates the presence of Adenine; C indicates the presenceof Cytosine; DNA is Deoxyribonucleic acid; G indicates the presence ofGuanine; RNA is Ribonucleic acid; T indicates the presence of Thymine;and U indicates the presence of Uracil. In embodiments, each of thefollowing terms shall have the definition set forth below A—Adenine;C—Cytosine; DNA—Deoxyribonucleic acid; G—Guanine; RNA—Ribonucleic acid;T—Thymine; and U—Uracil.

All embodiments of U.S. Pat. No. 6,664,079 (the contents of which arehereby incorporated by reference) with regard to sequencing a nucleicacid are specifically envisioned here.

“Alkyldithiomethyl” refers to a compound or moiety or portion thereof,comprising a dithio group, where one of the sulfurs is directlyconnected to a methyl group (i.e., a methylene linking group) and theother sulfur is directly connected to an alkyl group. An example is thestructure

wherein R is an alkyl group (e.g., methyl or ethyl) and the wavy line(e.g.,

) represents a point of connection to another portion of the compound.In some cases, the alkyldithiomethyl is methyldithiomethyl,ethyldithiomethyl, propyldithiomethyl, isopropyldithiomethyl,butyldithiomethyl, t-butyldithiomethyl, or phenyldithiomethyl.

The term “protecting group” is used in accordance with its ordinarymeaning in organic chemistry and refers to a moiety covalently bound toa heteroatom, heterocycloalkyl, or heteroaryl to prevent reactivity ofthe heteroatom, heterocycloalkyl, or heteroaryl during one or morechemical reactions performed prior to removal of the protecting group.Typically a protecting group is bound to a heteroatom (e.g., O) during apart of a multipart synthesis wherein it is not desired to have theheteroatom react (e.g., a chemical reduction) with the reagent.Following protection the protecting group may be removed (e.g., bymodulating the pH). In embodiments the protecting group is an alcoholprotecting group. Non-limiting examples of alcohol protecting groupsinclude acetyl, benzoyl, benzyl, methoxymethyl ether (MOM),tetrahydropyranyl (THP), and silyl ether (e.g., trimethylsilyl (TMS)).In embodiments the protecting group is an amine protecting group.Non-limiting examples of amine protecting groups include carbobenzyloxy(Cbz), tert-butyloxycarbonyl (BOC), 9-Fluorenylmethyloxycarbonyl (FMOC),acetyl, benzoyl, benzyl, carbamate, p-methoxybenzyl ether (PMB), andtosyl (Ts). In embodiments, the protecting group is a nucleosideprotecting group. In embodiments, the protecting group is a5′-O-nucleoside protecting group.

The term “5′-nucleoside protecting group” as used herein refers to amoiety covalently bound to a heteroatom (e.g., O) on the 5′ position ofsugar to prevent reactivity of the heteroatom during one or morechemical reactions performed prior to removal of the protecting group.Typically a protecting group is bound to a heteroatom (e.g., O) during apart of a multipart synthesis wherein it is not desired to have theheteroatom react (e.g., during a chemical reduction) with the reagent.Following protection the protecting group may be removed by anyappropriate means (e.g., by modulating the pH). Non-limiting examples of5′-O-nucleoside protecting groups include silyl ethers (e.g.,tert-butyl-diphenylsilyl (TBDPS), or primary and secondarytert-butyldimethylsilyl (TBDMS)) or trityl (e.g., 4,4′-dimethoxytrityl(DMT)). In embodiments, R¹ includes a protecting group found in Green'sProtective Groups in Organic Chemistry, Wiley, Fourth edition, 2007,Peter G. M. Wuts and Theodora W. Greene, and Current Protocols inNucleic Acid Chemistry (2000) 2.3.1-2.3.34, John Wiley & Sons, Inc.which is incorporated herein by reference in its entirety for allpurposes.

The term “deprotect” or “deprotecting” is used in accordance with itsordinary meaning in organic chemistry and refers a process or chemicalreaction that remove a protecting group, which is covalently bound to aheteroatom, heterocycloalkyl, or heteroaryl, to recover reactivity ofthe heteroatom, heterocycloalkyl, or heteroaryl for subsequent chemicalreactions or metabolic pathway. The “deprotecting agent” or“deprotecting reagent” is used in accordance with its ordinary meaningin organic chemistry and refers to a molecule used for deprotecting. Inembodiments, the deprotecting agent is an acid or a base. Inembodiments, the deprotecting agent includes alpha-hydroxy amines (aminoalcohol), primary amines and secondary amines. In embodiments, thedeprotecting agent is ammonium salt (e.g., ammonium hydroxide, ammoniumhydrogen sulfate, ceric ammonium nitrate, or ammonium fluoride). Inembodiments, the deprotecting agent is concentrated ammonium hydroxide.The terms “5′-nucleoside protecting group” and “5′-O-nucleosideprotecting group” are used interchangeably herein.

The term “reaction vessel” is used in accordance with its ordinarymeaning in chemistry or chemical engineering, and refers to a containerhaving an inner volume in which a reaction takes place. In embodiments,the reaction vessel may be designed to provide suitable reactionconditions such as reaction volume, reaction temperature or pressure,and stirring or agitation, which may be adjusted to ensure that thereaction proceeds with a desired, sufficient or highest efficiency forproducing a product from the chemical reaction. In embodiments, thereaction vessel is a container for liquid, gas or solid. In embodiments,the reaction vessel may include an inlet, an outlet, a reservoir and thelike. In embodiments, the reaction vessel is connected to a pump (e.g.,vacuum pump), a controller (e.g., CPU), or a monitoring device (e.g., UVdetector or spectrophotometer). In embodiments, the reaction vessel is aflow cell. In embodiments, the reaction vessel is within a sequencingdevice.

A person of ordinary skill in the art will understand when a variable(e.g., moiety or linker) of a compound or of a compound genus (e.g., agenus described herein) is described by a name or formula of astandalone compound with all valencies filled, the unfilled valence(s)of the variable will be dictated by the context in which the variable isused. For example, when a variable of a compound as described herein isconnected (e.g., bonded) to the remainder of the compound through asingle bond, that variable is understood to represent a monovalent form(i.e., capable of forming a single bond due to an unfilled valence) of astandalone compound (e.g., if the variable is named “methane” in anembodiment but the variable is known to be attached by a single bond tothe remainder of the compound, a person of ordinary skill in the artwould understand that the variable is actually a monovalent form ofmethane, i.e., methyl or —CH₃). Likewise, for a linker variable (e.g.,L¹, L², or L³ as described herein), a person of ordinary skill in theart will understand that the variable is the divalent form of astandalone compound (e.g., if the variable is assigned to “PEG” or“polyethylene glycol” in an embodiment but the variable is connected bytwo separate bonds to the remainder of the compound, a person ofordinary skill in the art would understand that the variable is adivalent (i.e., capable of forming two bonds through two unfilledvalences) form of PEG instead of the standalone compound PEG).

As used herein, the term “kit” refers to any delivery system fordelivering materials. In the context of reaction assays, such deliverysystems include systems that allow for the storage, transport, ordelivery of reaction reagents (e.g., oligonucleotides, enzymes, etc. inthe appropriate containers) and/or supporting materials (e.g.,packaging, buffers, written instructions for performing a method, etc.)from one location to another. For example, kits include one or moreenclosures (e.g., boxes) containing the relevant reaction reagentsand/or supporting materials. As used herein, the term “fragmented kit”refers to a delivery system comprising two or more separate containersthat each contain a subportion of the total kit components. Thecontainers may be delivered to the intended recipient together orseparately. For example, a first container may contain an enzyme for usein an assay, while a second container contains oligonucleotides. Incontrast, a “combined kit” refers to a delivery system containing all ofthe components of a reaction assay in a single container (e.g., in asingle box housing each of the desired components). The term “kit”includes both fragmented and combined kits.

As used herein, the terms “sequencing”, “sequence determination”,“determining a nucleotide sequence”, and the like include determinationof a partial or complete sequence information, including theidentification, ordering, or locations of the nucleotides that comprisethe polynucleotide being sequenced, and inclusive of the physicalprocesses for generating such sequence information. That is, the termincludes sequence comparisons, consensus sequence determination, contigassembly, fingerprinting, and like levels of information about a targetpolynucleotide, as well as the express identification and ordering ofnucleotides in a target polynucleotide. The term also includes thedetermination of the identification, ordering, and locations of one,two, or three of the four types of nucleotides within a targetpolynucleotide. In some embodiments, a sequencing process describedherein comprises contacting a template and an annealed primer with asuitable polymerase under conditions suitable for polymerase extensionand/or sequencing. The sequencing methods are preferably carried outwith the target polynucleotide arrayed on a solid substrate. Multipletarget polynucleotides can be immobilized on the solid support throughlinker molecules, or can be attached to particles, e.g., microspheres,which can also be attached to a solid substrate. In embodiments, thesolid substrate is in the form of a chip, a bead, a well, a capillarytube, a slide, a wafer, a filter, a fiber, a porous media, or a column.In embodiments, the solid substrate is gold, quartz, silica, plastic,glass, diamond, silver, metal, or polypropylene. In embodiments, thesolid substrate is porous.

As used herein, the term “extension” or “elongation” is used inaccordance with its plain and ordinary meanings and refer to synthesisby a polymerase of a new polynucleotide strand complementary to atemplate strand by adding free nucleotides (e.g., dNTPs) from a reactionmixture that are complementary to the template in the 5′-to-3′direction. Extension includes condensing the 5′-phosphate group of thedNTPs with the 3′-hydroxy group at the end of the nascent (elongating)polynucleotide strand.

As used herein, the term “sequencing read” is used in accordance withits plain and ordinary meaning and refers to an inferred sequence ofnucleotide bases (or nucleotide base probabilities) corresponding to allor part of a single polynucleotide fragment. A sequencing read mayinclude 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or morenucleotide bases. In embodiments, a sequencing read includes reading abarcode sequence and a template nucleotide sequence. In embodiments, asequencing read includes reading a template nucleotide sequence. Inembodiments, a sequencing read includes reading a barcode and not atemplate nucleotide sequence.

As used herein, the term “barcode sequence” (which may be referred to asa “tag,” a “molecular barcode,” a “molecular identifier,” an “identifiersequence,” or a “unique molecular identifier (UMI)”) refers to anymaterial (e.g., a nucleotide sequence, a nucleic acid molecule feature)that is capable of distinguishing an individual molecule in a largeheterogeneous population of molecules. Generally, a barcode sequence isunique in a pool of barcode sequences that differ from one another insequence, or is uniquely associated with a particular samplepolynucleotide in a pool of sample polynucleotides. In embodiments,barcode sequences are about or at least about 5, 6, 7, 8, 9, 10, 15, 20,25, 30, 40, 50, 75 or more nucleotides in length. In embodiments,barcode sequences are shorter than 20, 15, 10, 9, 8, 7, 6, or 5nucleotides in length. In embodiments, barcode sequences are about 10 toabout 50 nucleotides in length, such as about 15 to about 40 or about 20to about 30 nucleotides in length. In a pool of different barcodesequences, barcode sequences may have the same or different lengths. Ingeneral, barcode sequences are of sufficient length and includesequences that are sufficiently different to allow the identification ofsequencing reads that originate from the same sample polynucleotidemolecule. In embodiments, each barcode sequence in a plurality ofbarcode sequences differs from every other barcode sequence in theplurality by at least three nucleotide positions, such as at least 3, 4,5, 6, 7, 8, 9, 10, or more nucleotide positions. In some embodiments,substantially degenerate barcode sequences may be known as random. Insome embodiments, a barcode sequence may include a nucleic acid sequencefrom within a pool of known sequences. In some embodiments, the barcodesequences may be pre-defined.

II. Compounds, Complexes, and Kits

In an aspect is provided a compound having the formula:

B¹ is a nucleobase. R⁷ is substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl. R⁸ issubstituted or unsubstituted alkyl. R¹ is independently hydrogen,halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂, —CHBr₂, —CHF₂, —CHI₂, —CH₂Cl,—CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H,—SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H,—NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃, —OCF₃, —OCBr₃, —OCI₃, —OCHCl₂,—OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl, —OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, a 5′-O-nucleoside protectinggroup, monophosphate moiety, polyphosphate moiety, or nucleic acidmoiety. R² is independently hydrogen, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃,—CHCl₂, —CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl; or apolymerase-compatible cleavable moiety.

In an aspect is provided a compound having the formula.

B¹ is a nucleobase. R¹ is independently hydrogen, halogen, —CCl₃, —CBr₃,—CF₃, —CI₃, —CHCl₂, —CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I,—CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCCl₃, —OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl, a5′-O-nucleoside protecting group, monophosphate moiety, polyphosphatemoiety, or nucleic acid moiety. R² is independently hydrogen, halogen,—CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂, —CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br,—CH₂F, —CH₂I, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H,—SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H,—NHC(O)OH, —NHOH, —OCCl₃, —OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂,—OCHI₂, —OCHF₂, —OCH₂Cl, —OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, substitutedor unsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl; or a polymerase-compatible cleavable moiety.

In an aspect is provided a compound having the formula:

B¹ is a nucleobase. R⁷ is substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl. R¹ isindependently hydrogen, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂,—CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl, a5′-O-nucleoside protecting group, monophosphate moiety, polyphosphatemoiety, or nucleic acid moiety. R² is independently hydrogen, halogen,—CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂, —CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br,—CH₂F, —CH₂I, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H,—SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H,—NHC(O)OH, —NHOH, —OCCl₃, —OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂,—OCHI₂, —OCHF₂, —OCH₂Cl, —OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, substitutedor unsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl; or a polymerase-compatible cleavable moiety.

In embodiments, the compounds of Formula I, Formula II, and Formula IIIare referred to as nucleotides. In embodiments, the compounds of FormulaI, Formula II, and Formula III include a nucleotide portion and a3′-O-reversible terminator. For example, the nucleotide portion is

and the 3′-O-reversible terminator portion is

for Formula I, II, and III respectively.

In embodiments, R¹ is —OH, a 5′-O-nucleoside protecting group,monophosphate moiety, polyphosphate moiety, or nucleic acid moiety. Inembodiments, R¹ is a triphosphate moiety. In embodiments, R¹ is —OH. Inembodiments, R¹ is a 5′-O-nucleoside protecting group. In embodiments,R¹ is a nucleic acid moiety. In embodiments, R¹ is independently amonophosphate moiety or a derivative thereof (e.g., including aphosphoramidate moiety, phosphorothioate moiety, phosphorodithioatemoiety, or O-methylphosphoroamidite moiety), polyphosphate moiety orderivative thereof (e.g., including a phosphoramidate, phosphorothioate,phosphorodithioate, or O-methylphosphoroamidite), or nucleic acid moietyor derivative thereof (e.g., including a phosphoramidate,phosphorothioate, phosphorodithioate, or O-methylphosphoroamidite).

In embodiments, R¹ is independently hydrogen, halogen, —CCl₃, —CBr₃,—CF₃, —CI₃, —CHCl₂, —CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I,—CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCCl₃, —OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl, a5′-O-nucleoside protecting group, monophosphate moiety, polyphosphatemoiety, or nucleic acid moiety. In embodiments, R¹ is independently amonophosphate moiety including a phosphodiester derivative. Inembodiments, R¹ is independently a polyphosphate moiety including aphosphodiester derivative. In embodiments, R¹ is independently a nucleicacid moiety including a phosphodiester derivative. In embodiments, R¹ isindependently a phosphoramidate moiety. In embodiments, R¹ isindependently a polyphosphate moiety including a phosphoramidate. Inembodiments, R¹ is independently a nucleic acid moiety including aphosphoramidate. In embodiments, R¹ is independently a phosphorothioatemoiety. In embodiments, R¹ is independently a polyphosphate moietyincluding a phosphorothioate. In embodiments, R¹ is independently anucleic acid moiety including a phosphorothioate. In embodiments, R¹ isindependently a phosphorodithioate moiety. In embodiments, R¹ isindependently a polyphosphate moiety including a phosphorodithioate. Inembodiments, R¹ is independently a nucleic acid moiety including aphosphorodithioate. In embodiments, R¹ is independently anO-methylphosphoroamidite moiety. In embodiments, R¹ is independently apolyphosphate moiety including an O-methylphosphoroamidite. Inembodiments, R¹ is independently a nucleic acid moiety including anO-methylphosphoroamidite. In embodiments, R¹ is independently a nucleicacid moiety including a nucleotide analog. In embodiments, R¹ isindependently a nucleic acid moiety including a plurality of optionallydifferent nucleotide analogs.

In embodiments, R¹ is independently a monophosphate moiety. Inembodiments, R¹ is independently a polyphosphate moiety. In embodiments,R¹ is independently a nucleic acid moiety. In embodiments, R¹ has theformula:

or ionized forms thereof. In embodiments, R¹ has the formula

or ionized forms thereof. In embodiments, R¹ has the formula

or ionized forms thereof. In embodiments, R¹ has the formula:

or ionized forms thereof, wherein np is an integer of 1 or greater. Inembodiments, np is an integer from 1 to 5. In embodiments, np is 1. Inembodiments, np is 2.

In embodiments, R¹ is independently a 5′-O-nucleoside protecting group,for example a 5′-O-nucleoside protecting group known in the art includethose described in Seliger H. Curr. Protoc Nucleic Acid Chem. 2001;Chapter 2 or K. Seio et al, Nucleic Acids Research Supplement 2, 27-28(2002); both of which are incorporated by reference for all purposes.Non-limiting examples of 5′-O-nucleoside protecting groups include2,2,2-Trichloroethyl carbonate (Troc), 2-Methoxyethoxymethyl ether(MEM), 2-Naphthylmethyl ether (Nap), 4-Methoxybenzyl ether (PMB),Acetate (Ac), Benzoate (Bz), Benzyl ether (Bn), Benzyloxymethyl acetal(BOM), Ethoxyethyl acetal (EE), Methoxymethyl acetal (MOM),Methoxypropyl acetal (MOP), Methyl ether, Tetrahydropyranyl acetal(THP), Triethylsilyl ether (TES), Triisopropylsilyl ether (TIPS),Trimethylsilyl ether (TMS), tert-Butyldimethylsilyl ether (TBS, TBDMS),or tert-Butyldiphenylsilyl ether (TBDPS). In embodiments, R¹ is

In embodiments, R¹ is independently hydrogen, halogen, —CCl₃, —CBr₃,—CF₃, —CI₃, —CHCl₂, —CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I,—CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₂NH₂, —NHNH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted alkyl (e.g., C₁-C₈, C₁-C₆, C₁-C₄, orC₁-C₂), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted heteroalkyl (e.g., 2 to 8, 2 to 6, 4 to 6, 2 to 3, or 4 to5 membered), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆),substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheterocycloalkyl (e.g., 3 to 8, 3 to 6, 4 to 6, 4 to 5, or 5 to 6membered), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted aryl (e.g., C₆-C₁₀ or phenyl), or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroaryl (e.g., 5 to 10membered, 5 to 9 membered, or 5 to 6 membered), or a 5′-O-nucleosideprotecting group; or R¹ is a monophosphate moiety, polyphosphate moiety,or nucleic acid moiety. In embodiments, R¹ is independently hydrogen,halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂, —CHBr₂, —CHF₂, —CHI₂, —CH₂Cl,—CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H,—SO₂NH₂, —NHNH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH,—NHOH, —OCCl₃, —OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂,—OCH₂Cl, —OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted alkyl (e.g., C₁-C₈, C₁-C₆,C₁-C₄, or C₁-C₂), substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroalkyl (e.g., 2 to 8, 2 to 6, 4 to 6, 2 to 3, or 4 to5 membered), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆),substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheterocycloalkyl (e.g., 3 to 8, 3 to 6, 4 to 6, 4 to 5, or 5 to 6membered), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted aryl (e.g., C₆-C₁₀ or phenyl), or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroaryl (e.g., 5 to 10membered, 5 to 9 membered, or 5 to 6 membered). In embodiments, R¹ is a5′-O-nucleoside protecting group. In embodiments, R¹ is a monophosphatemoiety, polyphosphate moiety, or nucleic acid moiety. In embodiments, R¹is a monophosphate moiety. In embodiments, R¹ is a polyphosphate moiety.In embodiments, R¹ is a nucleic acid moiety. In embodiments, R¹ ishydrogen. In embodiments, R¹ is a triphosphate moiety. In embodiments,R¹ is —OH.

In embodiments, a substituted R¹ (e.g., substituted alkyl, substitutedheteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,substituted aryl, and/or substituted heteroaryl) is substituted with atleast one substituent group, size-limited substituent group, or lowersubstituent group; wherein if the substituted R¹ is substituted with aplurality of groups selected from substituent groups, size-limitedsubstituent groups, and lower substituent groups; each substituentgroup, size-limited substituent group, and/or lower substituent groupmay optionally be different. In embodiments, when R¹ is substituted, itis substituted with at least one substituent group. In embodiments, whenR¹ is substituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when R¹ is substituted, it issubstituted with at least one lower substituent group. In embodiments,when R¹ is substituted, it is substituted with 1 to 10 substituentgroups. In embodiments, when R¹ is substituted, it is substituted with 1to 10 size-limited substituent groups. In embodiments, when R¹ issubstituted, it is substituted with 1 to 10 lower substituent groups. Inembodiments, when R¹ is substituted, it is substituted with 1 to 5substituent groups. In embodiments, when R¹ is substituted, it issubstituted with 1 to 5 size-limited substituent groups. In embodiments,when R¹ is substituted, it is substituted with 1 to 5 lower substituentgroups. In embodiments, when R¹ is substituted, it is substituted with asubstituent group. In embodiments, when R¹ is substituted, it issubstituted with a size-limited substituent group. In embodiments, whenR¹ is substituted, it is substituted with a lower substituent group.

In embodiments, R² is independently hydrogen, halogen, —CCl₃, —CBr₃,—CF₃, —CI₃, —CHCl₂, —CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I,—CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCCl₃, —OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl; or apolymerase-compatible cleavable moiety. In embodiments, R² is hydrogen.In embodiments, R² is —OH. In embodiments, R² is an—O-polymerase-compatible cleavable moiety, wherein the —O— is attachedto the 2′ position of the ribose sugar of a nucleotide and apolymerase-compatible cleavable moiety is as described herein.

In embodiments, R² is independently hydrogen, halogen, —CCl₃, —CBr₃,—CF₃, —CI₃, —CHCl₂, —CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I,—CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCCl₃, —OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, R^(2A)-substituted or unsubstitutedalkyl (e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄),R^(2A)-substituted or unsubstituted heteroalkyl (e.g., 2 to 20, 8 to 20,2 to 10, 2 to 8, 2 to 6, or 2 to 4 membered), R^(2A)-substituted orunsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, or C₅-C₆),R^(2A)-substituted or unsubstituted heterocycloalkyl (e.g., 3 to 8, 3 to6, or 5 to 6 membered), R^(2A)-substituted or unsubstituted aryl (e.g.,C₆-C₁₀, C₁₀, or phenyl), or R^(2A)-substituted or unsubstitutedheteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered). In embodiments,R² is independently hydrogen, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂,—CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, substituted or unsubstituted alkyl(e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄), substituted orunsubstituted heteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to6, or 2 to 4 membered), substituted or unsubstituted cycloalkyl (e.g.,C₃-C₈, C₃-C₆, or C₅-C₆), substituted or unsubstituted heterocycloalkyl(e.g., 3 to 8, 3 to 6, or 5 to 6 membered), substituted or unsubstitutedaryl (e.g., C₆-C₁₀, C₁₀, or phenyl), or substituted or unsubstitutedheteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered); or apolymerase-compatible cleavable moiety.

R^(2A) is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂,—CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, —NH₃ ⁺, —SO₃ ⁻, —OPO₃H⁻, —SCN,—ONO₂, R^(2B)-substituted or unsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀,C₁-C₈, C₁-C₆, or C₁-C₄), R^(2B)-substituted or unsubstituted heteroalkyl(e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to 6, or 2 to 4 membered),R^(2B)-substituted or unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, orC₅-C₆), R^(2B)-substituted or unsubstituted heterocycloalkyl (e.g., 3 to8, 3 to 6, or 5 to 6 membered), R^(2B)-substituted or unsubstituted aryl(e.g., C₆-C₁₀, C₁₀, or phenyl), or R^(2B)-substituted or unsubstitutedheteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered), or apolymerase-compatible cleavable moiety. In embodiments, R^(2A) isindependently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂, —CHBr₂,—CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂, —COOH,—CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂,—NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃, —OCF₃, —OCBr₃,—OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl, —OCH₂Br, —OCH₂I,—OCH₂F, —N₃, —SF₅, —NH₃ ⁺, —SO₃ ⁻, —OPO₃H⁻, —SCN, —ONO₂,R^(2B)-substituted or unsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₈,C₁-C₆, or C₁-C₄), R^(2B)-substituted or unsubstituted heteroalkyl (e.g.,2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to 6, or 2 to 4 membered),R^(2B)-substituted or unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, orC₅-C₆), R^(2B)-substituted or unsubstituted heterocycloalkyl (e.g., 3 to8, 3 to 6, or 5 to 6 membered), R^(2B)-substituted or unsubstituted aryl(e.g., C₆-C₁₀, C_(Io), or phenyl), or R^(2B)-substituted orunsubstituted heteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered). Inembodiments, R^(2A) is independently a polymerase-compatible cleavablemoiety.

R^(2B) is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂,—CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, —NH₃ ⁺, —SO₃ ⁻, —OPO₃H⁻, —SCN,—ONO₂, R^(2C)-substituted or unsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀,C₁-C₈, C₁-C₆, or C₁-C₄), R^(2C)-substituted or unsubstituted heteroalkyl(e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to 6, or 2 to 4 membered),R^(2C)-substituted or unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, orC₅-C₆), R^(2C)-substituted or unsubstituted heterocycloalkyl (e.g., 3 to8, 3 to 6, or 5 to 6 membered), R^(2C)-substituted or unsubstituted aryl(e.g., C₆-C₁₀, C₁₀, or phenyl), or R^(2C)-substituted or unsubstitutedheteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered).

R^(2C) is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂,—CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, —NH₃ ⁺, —SO₃ ⁻, —OPO₃H⁻, —SCN,—ONO₂, unsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, orC₁-C₄), unsubstituted heteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to8, 2 to 6, or 2 to 4 membered), unsubstituted cycloalkyl (e.g., C₃-C₈,C₃-C₆, or C₅-C₆), unsubstituted heterocycloalkyl (e.g., 3 to 8, 3 to 6,or 5 to 6 membered), unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, or phenyl),or unsubstituted heteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered).

In embodiments, R² is hydrogen. In embodiments, R² is —OH. Inembodiments, R² is —O-polymerase-compatible cleavable moiety.

In embodiments, R² is a polymerase-compatible cleavable moiety or an—O-polymerase-compatible cleavable moiety; and the polymerase-compatiblecleavable moiety is

R^(5A) is independently hydrogen, halogen, —CX^(5A) ₃, —CHX^(5A) ₂,—CH₂X^(5A), —OCX^(5A) ₃, —OCH₂X^(5A), —OCHX^(5A) ₂, —CN, —OH, —SH, —NH₂,—COOH, —CONH₂, —NO₂, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂,—NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —N₃, —SF₅, —NH₃ ⁺, —SO₃⁻, —OPO₃H⁻, —SCN, —ONO₂, substituted or unsubstituted alkyl (e.g.,C₁-C₈, C₁-C₆, C₁-C₄, or C₁-C₂), substituted or unsubstituted heteroalkyl(e.g., 2 to 8, 2 to 6, 4 to 6, 2 to 3, or 4 to 5 membered), substitutedor unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆),substituted or unsubstituted heterocycloalkyl (e.g., 3 to 8, 3 to 6, 4to 6, 4 to 5, or 5 to 6 membered), substituted or unsubstituted aryl(e.g., C₆-C₁₀ or phenyl), or substituted or unsubstituted heteroaryl(e.g., 5 to 10, 5 to 9, or 5 to 6 membered). R^(5B) is independentlyhydrogen, halogen, —CX^(5B) ₃, —CHX^(5B) ₂, —CH₂X^(5B), —OCX^(5B) ₃,—OCH₂X^(5B), —OCHX^(5B) ₂, —CN, —OH, —SH, —NH₂, —COOH, —CONH₂, —NO₂,—SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H,—NHC(O)H, —NHC(O)OH, —NHOH, —N₃, —SF₅, —NH₃ ⁺, —SO₃ ⁻, —OPO₃H⁻, —SCN,—ONO₂, substituted or unsubstituted alkyl (e.g., C₁-C₈, C₁-C₆, C₁-C₄, orC₁-C₂), substituted or unsubstituted heteroalkyl (e.g., 2 to 8, 2 to 6,4 to 6, 2 to 3, or 4 to 5 membered), substituted or unsubstitutedcycloalkyl (e.g., C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆), substituted orunsubstituted heterocycloalkyl (e.g., 3 to 8, 3 to 6, 4 to 6, 4 to 5, or5 to 6 membered), substituted or unsubstituted aryl (e.g., C₆-C₁₀ orphenyl), or substituted or unsubstituted heteroaryl (e.g., 5 to 10, 5 to9, or 5 to 6 membered). In embodiments, R5^(A) and R5^(B) are combinedto form an oxo. R5^(c) is hydrogen, halogen, —CX^(5C) ₃, —CHX^(5C) ₂,—CH₂X^(5C), —OCX^(5C) ₃, —OCH₂X^(5C), —OCHX^(5C) ₂, —CN, —OH, —SH, —NH₂,—COOH, —CONH₂, —NO₂, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂,—NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —N₃, —SF₅, —NH₃ ⁺, —SO₃⁻, —OPO₃H⁻, —SCN, —ONO₂, substituted or unsubstituted alkyl (e.g.,C₁-C₈, C₁-C₆, C₁-C₄, or C₁-C₂), substituted or unsubstituted heteroalkyl(e.g., 2 to 8, 2 to 6, 4 to 6, 2 to 3, or 4 to 5 membered), substitutedor unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆),substituted or unsubstituted heterocycloalkyl (e.g., 3 to 8, 3 to 6, 4to 6, 4 to 5, or 5 to 6 membered), substituted or unsubstituted aryl(e.g., C₆-C₁₀ or phenyl), or substituted or unsubstituted heteroaryl(e.g., 5 to 10, 5 to 9, or 5 to 6 membered). In embodiments, R^(5C) isunsubstituted C₁-C₄ alkyl. In embodiments, R^(5C) is unsubstitutedmethyl. In embodiments, R^(5C) is unsubstituted tert-butyl. The symbolsX^(5A), X^(5B), and X^(5C) are independently —F, —Cl, —Br, or —I.

In embodiments, the polymerase-compatible cleavable moiety is

R^(5A), R^(5B) and R^(5C) are as described herein, including inembodiments. In embodiments, R² is an —O-polymerase-compatible cleavablemoiety; and the polymerase-compatible cleavable moiety is

In embodiments, R² is a polymerase-compatible cleavable moiety or an—O-polymerase-compatible cleavable moiety; and the polymerase-compatiblecleavable moiety is

In embodiments, R^(5A) is independently hydrogen, halogen, —CX^(5A) ₃,—CHX^(5A) ₂, —CH₂X^(5A), —OCX^(5A) ₃, —OCH₂X^(5A), —OCHX^(5A) ₂, —CN,—OH, —SH, —NH₂, —COOH, —CONH₂, —NO₂, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—N₃, —SF₅, —NH₃ ⁺, —SO₃ ⁻, —OPO₃H⁻, —SCN, —ONO₂, R^(5D)-substituted orunsubstituted alkyl, R^(5D)-substituted or unsubstituted heteroalkyl,R^(5D)-substituted or unsubstituted cycloalkyl, R^(5D)-substituted orunsubstituted heterocycloalkyl, R^(5D)-substituted or unsubstitutedaryl, or R^(5D)-substituted or unsubstituted heteroaryl. R^(5D) isindependently halogen, oxo, —CX^(5D) ₃, —CHX^(5D) ₂, —CH₂X^(5D),—OCX^(5D) ₃, —OCH₂X^(5D), —OCHX^(5D) ₂, —CN, —OH, —SH, —NH₂, —COOH,—CONH₂, —NO₂, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂,—NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —N₃, —SF₅, —NH₃ ⁺, —SO₃⁻, —OPO₃H⁻, —SCN, —ONO₂, R^(5E)-substituted or unsubstituted alkyl,R^(5E)-substituted or unsubstituted heteroalkyl, R^(5E)-substituted orunsubstituted cycloalkyl, R^(5E)-substituted or unsubstitutedheterocycloalkyl, R^(5E)-substituted or unsubstituted aryl, orR^(5E)-substituted or unsubstituted heteroaryl. R^(5E) is independentlyhalogen, oxo, —CX^(5E) ₃, —CHX^(5E) ₂, —CH₂X^(5E), —OCX^(5E) ₃,—OCH₂X^(5E), —OCHX^(5E) ₂, —CN, —OH, —SH, —NH₂, —COOH, —CONH₂, —NO₂,—SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H,—NHC(O)H, —NHC(O)OH, —NHOH, —N₃, —SF₅, —NH₃ ⁺, —SO₃ ⁻, —OPO₃H⁻, —SCN,—ONO₂, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstitutedcycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, orunsubstituted heteroaryl. In embodiments, R^(5B) is independentlyhydrogen, halogen, —CX^(5B) ₃, —CHX^(5B) ₂, —CH₂X^(5B), —OCX^(5B) ₃,—OCH₂X^(5B), —OCHX^(5B) ₂, —CN, —OH, —SH, —NH₂, —COOH, —CONH₂, —NO₂,—SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H,—NHC(O)H, —NHC(O)OH, —NHOH, —N₃, —SF₅, —NH₃ ⁺, —SO₃—, —OPO₃H⁻, —SCN,—ONO₂, R^(5F)-substituted or unsubstituted alkyl, R^(5F)-substituted orunsubstituted heteroalkyl, R^(5F)-substituted or unsubstitutedcycloalkyl, R^(5F)-substituted or unsubstituted heterocycloalkyl,R^(5F)-substituted or unsubstituted aryl, or R^(5F)-substituted orunsubstituted heteroaryl. R^(5F) is independently halogen, oxo, —CX^(5F)₃, —CHX^(5F) ₂, —CH₂X^(5F), —OCX^(5F) ₃, —OCH₂X^(5F), —OCHX^(5F) ₂, —CN,—OH, —SH, —NH₂, —COOH, —CONH₂, —NO₂, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—N₃, —SF₅, —NH₃ ⁺, —SO₃ ⁻, —OPO₃H, —SCN, —ONO₂, R^(5G)-substituted orunsubstituted alkyl, R^(5G)-substituted or unsubstituted heteroalkyl,R^(5G)-substituted or unsubstituted cycloalkyl, R^(5G)-substituted orunsubstituted heterocycloalkyl, R^(5G)-substituted or unsubstitutedaryl, or R^(5G)-substituted or unsubstituted heteroaryl. R^(5G) isindependently halogen, oxo, —CX^(5G) ₃, —CHX^(5G) ₂, —CH₂X^(5G),—OCX^(5G) ₃, —OCH₂X^(5G), —OCHX^(5G) ₂, —CN, —OH, —SH, —NH₂, —COOH,—CONH₂, —NO₂, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂,—NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —N₃, —SF₅, —NH₃ ⁺, —SO₃⁻, —OPO₃H⁻, —SCN, —ONO₂, unsubstituted alkyl, unsubstituted heteroalkyl,unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstitutedaryl, or unsubstituted heteroaryl. In embodiments, R^(5A) and R^(5B) arecombined to form an oxo. In embodiments, R^(5C) is independentlyhydrogen, halogen, —CX^(5C) ₃, —CHX^(5C) ₂, —CH₂X^(5C), —OCX^(5C) ₃,—OCH₂X^(5C), —OCHX^(5C) ₂, —CN, —OH, —SH, —NH₂, —COOH, —CONH₂, —NO₂,—SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H,—NHC(O)H, —NHC(O)OH, —NHOH, —N₃, —SF₅, —NH₃ ⁺, —SO₃ ⁻, —OPO₃H⁻, —SCN,—ONO₂, R^(5H)-substituted or unsubstituted alkyl, R^(5H)-substituted orunsubstituted heteroalkyl, R^(5H)-substituted or unsubstitutedcycloalkyl, R^(5H)-substituted or unsubstituted heterocycloalkyl,R^(5H)-substituted or unsubstituted aryl, or R^(5H)-substituted orunsubstituted heteroaryl. R^(5H) is independently halogen, oxo, —CX^(5H)₃, —CHX^(5H) ₂, —CH₂X^(5H), —OCX^(5H) ₃, —OCH₂X^(5H), —OCHX^(5H) ₂, —CN,—OH, —SH, —NH₂, —COOH, —CONH₂, —NO₂, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—N₃, —SF₅, —NH₃ ⁺, —SO₃—, —OPO₃H, —SCN, —ONO₂, R^(5I)-substituted orunsubstituted alkyl, R^(5I)-substituted or unsubstituted heteroalkyl,R^(5I)-substituted or unsubstituted cycloalkyl, R^(5I)-substituted orunsubstituted heterocycloalkyl, R^(5I)-substituted or unsubstitutedaryl, or R^(5I)-substituted or unsubstituted heteroaryl. R^(5I) isindependently halogen, oxo, —CX^(5I) ₃, —CHX^(5I) ₂, —CH₂X^(5I),—OCX^(5I) ₃, —OCH₂X^(5I), —OCHX^(5I) ₂, —CN, —OH, —SH, —NH₂, —COOH,—CONH₂, —NO₂, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂,—NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —N₃, —SF₅, —NH₃ ⁺, —SO₃⁻, —OPO₃H⁻, —SCN, —ONO₂, unsubstituted alkyl, unsubstituted heteroalkyl,unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstitutedaryl, or unsubstituted heteroaryl. In embodiments, R^(5C) isunsubstituted C₁-C₄ alkyl. In embodiments, R^(5C) is unsubstitutedmethyl. In embodiments, R^(5C) is unsubstituted tert-butyl. The symbolsX^(5A), X^(5B), X^(5C), X^(5D), X^(5E), X^(5F), X^(5G), X^(5H), andX^(5I) are independently —F, —Cl, —Br, or —I.

In embodiments, R² is a polymerase-compatible cleavable moiety or an—O-polymerase-compatible cleavable moiety; and the polymerase-compatiblecleavable moiety is

In embodiments, R^(5A) is independently hydrogen, halogen, —CX^(5A) ₃,—CHX^(5A) ₂, —CH₂X^(5A), —OCX^(5A) ₃, —OCH₂X^(5A), —OCHX^(5A) ₂, —CN,—OH, —SH, —NH₂, —COOH, —CONH₂, —NO₂, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—N₃, —SF₅, —NH₃ ⁺, —SO₃—, —OPO₃H⁻, —SCN, —ONO₂, R^(5D)-substituted orunsubstituted alkyl (e.g., C₁-C₈, C₁-C₆, C₁-C₄, or C₁—C₂),R^(5D)-substituted or unsubstituted heteroalkyl (e.g., 2 to 8, 2 to 6, 4to 6, 2 to 3, or 4 to 5 membered), R^(5D)-substituted or unsubstitutedcycloalkyl (e.g., C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆), R^(5D)-substituted orunsubstituted heterocycloalkyl (e.g., 3 to 8, 3 to 6, 4 to 6, 4 to 5, or5 to 6 membered), R^(5D)-substituted or unsubstituted aryl (e.g., C₆-C₁₀or phenyl), or R^(5D)-substituted or unsubstituted heteroaryl (e.g., 5to 10, 5 to 9, or 5 to 6 membered). In embodiments, R^(5D) isindependently halogen, oxo, —CX^(5D) ₃, —CHX^(5D) ₂, —CH₂X_(5D),—OCX^(5D) ₃, —OCH₂X^(5D), —OCHX^(5D) ₂, —CN, —OH, —SH, —NH₂, —COOH,—CONH₂, —NO₂, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂,—NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —N₃, —SF₅, —NH₃ ⁺, —SO₃⁻, —OPO₃H⁻, —SCN, —ONO₂, R^(5E)-substituted or unsubstituted alkyl(e.g., C₁-C₈, C₁-C₆, C₁-C₄, or C₁-C₂), R^(5E)-substituted orunsubstituted heteroalkyl (e.g., 2 to 8, 2 to 6, 4 to 6, 2 to 3, or 4 to5 membered), R^(5E)-substituted or unsubstituted cycloalkyl (e.g.,C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆), R^(5E)-substituted or unsubstitutedheterocycloalkyl (e.g., 3 to 8, 3 to 6, 4 to 6, 4 to 5, or 5 to 6membered), R^(5E)-substituted or unsubstituted aryl (e.g., C₆-C₁₀ orphenyl), or R^(5E)-substituted or unsubstituted heteroaryl (e.g., 5 to10, 5 to 9, or 5 to 6 membered). In embodiments, R^(5E) is independentlyhalogen, oxo, —CX^(5E) ₃, —CHX^(5E) ₂, —CH₂X^(5E), —OCX^(5E) ₃,—OCH₂X^(5E), —OCHX^(5E) ₂, —CN, —OH, —SH, —NH₂, —COOH, —CONH₂, —NO₂,—SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H,—NHC(O)H, —NHC(O)OH, —NHOH, —N₃, —SF₅, —NH₃ ⁺, —SO₃ ⁻, —OPO₃H⁻, —SCN,—ONO₂, unsubstituted alkyl (e.g., C₁-C₈, C₁-C₆, C₁-C₄, or C₁-C₂),unsubstituted heteroalkyl (e.g., 2 to 8, 2 to 6, 4 to 6, 2 to 3, or 4 to5 membered), unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, C₄-C₆, orC₅-C₆), unsubstituted heterocycloalkyl (e.g., 3 to 8, 3 to 6, 4 to 6, 4to 5, or 5 to 6 membered), unsubstituted aryl (e.g., C₆-C₁₀ or phenyl),or unsubstituted heteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered).In embodiments, R^(5B) is independently hydrogen, halogen, —CX^(5B) ₃,—CHX^(5B) ₂, —CH₂X^(5B), —OCX^(5B) ₃, —OCH₂X^(5B), —OCHX^(5B) ₂, —CN,—OH, —SH, —NH₂, —COOH, —CONH₂, —NO₂, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—N₃, —SF₅, —NH₃ ⁺, —SO₃ ⁻, —OPO₃H⁻, —SCN, —ONO₂, R^(5F)-substituted orunsubstituted alkyl (e.g., C₁-C₈, C₁-C₆, C₁-C₄, or C₁-C₂),R^(5F)-substituted or unsubstituted heteroalkyl (e.g., 2 to 8, 2 to 6, 4to 6, 2 to 3, or 4 to 5 membered), R^(5F)-substituted or unsubstitutedcycloalkyl (e.g., C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆), R^(5F)-substituted orunsubstituted heterocycloalkyl (e.g., 3 to 8, 3 to 6, 4 to 6, 4 to 5, or5 to 6 membered), R^(5F)-substituted or unsubstituted aryl (e.g., C₆-C₁₀or phenyl), or R^(5F)-substituted or unsubstituted heteroaryl (e.g., 5to 10, 5 to 9, or 5 to 6 membered). In embodiments, R5F is independentlyhalogen, oxo, —CX^(5F) ₃, —CHX^(5F) ₂, —CH₂X^(5F), —OCX^(5F) ₃,—OCH₂X^(5F), —OCHX^(5F) ₂, —CN, —OH, —SH, —NH₂, —COOH, —CONH₂, —NO₂,—SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H,—NHC(O)H, —NHC(O)OH, —NHOH, —N₃, —SF₅, —NH₃ ⁺, —SO₃ ⁻, —OPO₃H⁻, —SCN,—ONO₂, R^(5G)-substituted or unsubstituted alkyl (e.g., C₁-C₈, C₁-C₆,C₁-C₄, or C₁-C₂), R^(5G)-substituted or unsubstituted heteroalkyl (e.g.,2 to 8, 2 to 6, 4 to 6, 2 to 3, or 4 to 5 membered), R^(5G)-substitutedor unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆),R^(5G)-substituted or unsubstituted heterocycloalkyl (e.g., 3 to 8, 3 to6, 4 to 6, 4 to 5, or 5 to 6 membered), R^(5G)-substituted orunsubstituted aryl (e.g., C₆-C₁₀ or phenyl), or R^(5G)-substituted orunsubstituted heteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered). Inembodiments, R^(5G) is independently halogen, oxo, —CX^(5G) ₃, —CHX^(5G)₂, —CH₂X^(5G), —OCX^(5G) ₃, —OCH₂X^(5G), —OCHX^(5G) ₂, —CN, —OH, —SH,—NH₂, —COOH, —CONH₂, —NO₂, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —N₃,—SF₅, —NH₃ ⁺, —SO₃ ⁻, —OPO₃H⁻, —SCN, —ONO₂, unsubstituted alkyl (e.g.,C₁-C₈, C₁-C₆, C₁-C₄, or C₁-C₂), unsubstituted heteroalkyl (e.g., 2 to 8,2 to 6, 4 to 6, 2 to 3, or 4 to 5 membered), unsubstituted cycloalkyl(e.g., C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆), unsubstituted heterocycloalkyl(e.g., 3 to 8, 3 to 6, 4 to 6, 4 to 5, or 5 to 6 membered),unsubstituted aryl (e.g., C₆-C₁₀ or phenyl), or unsubstituted heteroaryl(e.g., 5 to 10, 5 to 9, or 5 to 6 membered). In embodiments, R^(5A) andR^(5B) are combined to form an oxo. In embodiments, R^(5C) isindependently hydrogen, halogen, —CX^(5C) ₃, —CHX^(5C) ₂, —CH₂X^(5C),—OCX^(5C) ₃, —OCH₂X^(5C), —OCHX^(5C) ₂, —CN, —OH, —SH, —NH₂, —COOH,—CONH₂, —NO₂, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂,—NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —N₃, —SF₅, —NH₃ ⁺,—SO₃—, —OPO₃H, —SCN, —ONO₂, R^(5H)-substituted or unsubstituted alkyl(e.g., C₁-C₈, C₁-C₆, C₁-C₄, or C₁-C₂), R^(5H)-substituted orunsubstituted heteroalkyl (e.g., 2 to 8, 2 to 6, 4 to 6, 2 to 3, or 4 to5 membered), R^(5H)-substituted or unsubstituted cycloalkyl (e.g.,C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆), R^(5H)-substituted or unsubstitutedheterocycloalkyl (e.g., 3 to 8, 3 to 6, 4 to 6, 4 to 5, or 5 to 6membered), R^(5H)-substituted or unsubstituted aryl (e.g., C₆-C₁₀ orphenyl), or R^(5H)-substituted or unsubstituted heteroaryl (e.g., 5 to10, 5 to 9, or 5 to 6 membered). In embodiments, R^(5H) is independentlyhalogen, oxo, —CX^(5H) ₃, —CHX^(5H) ₂, —CH₂X^(5H), —OCX^(5H) ₃,—OCH₂X^(5H), —OCHX^(5H) ₂, —CN, —OH, —SH, —NH₂, —COOH, —CONH₂, —NO₂,—SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H,—NHC(O)H, —NHC(O)OH, —NHOH, —N₃, —SF₅, —NH₃ ⁺, —SO₃ ⁻, —OPO₃H⁻, —SCN,—ONO₂, R^(5I)-substituted or unsubstituted alkyl (e.g., C₁-C₈, C₁-C₆,C₁-C₄, or C₁-C₂), R^(5I)-substituted or unsubstituted heteroalkyl (e.g.,2 to 8, 2 to 6, 4 to 6, 2 to 3, or 4 to 5 membered), R^(5I)-substitutedor unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆),R^(5I)-substituted or unsubstituted heterocycloalkyl (e.g., 3 to 8, 3 to6, 4 to 6, 4 to 5, or 5 to 6 membered), R^(5I)-substituted orunsubstituted aryl (e.g., C₆-C₁₀ or phenyl), or R^(5I)-substituted orunsubstituted heteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered). Inembodiments, R^(5I) is independently halogen, oxo, —CX^(5I) ₃, —CHX^(5I)₂, —CH₂X^(5I), —OCX^(5I) ₃, —OCH₂X^(5I), —OCHX^(5I) ₂, —CN, —OH, —SH,—NH₂, —COOH, —CONH₂, —NO₂, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —N₃,—SF₅, —NH₃ ⁺, —SO₃ ⁻, —OPO₃H⁻, —SCN, —ONO₂, unsubstituted alkyl (e.g.,C₁-C₈, C₁-C₆, C₁-C₄, or C₁-C₂), unsubstituted heteroalkyl (e.g., 2 to 8,2 to 6, 4 to 6, 2 to 3, or 4 to 5 membered), unsubstituted cycloalkyl(e.g., C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆), unsubstituted heterocycloalkyl(e.g., 3 to 8, 3 to 6, 4 to 6, 4 to 5, or 5 to 6 membered),unsubstituted aryl (e.g., C₆-C₁₀ or phenyl), or unsubstituted heteroaryl(e.g., 5 to 10, 5 to 9, or 5 to 6 membered). In embodiments, R^(5C) isunsubstituted C₁-C₄ alkyl. In embodiments, R^(5C) is unsubstitutedmethyl. In embodiments, R^(5C) is unsubstituted tert-butyl. The symbolsX^(5A), X^(5B), X^(5C), X^(5D), X^(5E), X^(5F), X^(5G), X⁵H, and X^(5I)are independently —F, —Cl, —Br, or —I.

In embodiments, R^(5A) is independently hydrogen, halogen, —CX^(5A) ₃,—CHX^(5A) ₂, —CH₂X^(5A), —OCX^(5A) ₃, —OCH₂X^(5A), —OCHX^(5A) ₂, —CN,—OH, —SH, —NH₂, —COOH, —CONH₂, —NO₂, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—N₃, —SF₅, —NH₃ ⁺, —SO₃—, —OPO₃H, —SCN, —ONO₂, R^(5D)-substituted C₁-C₄alkyl (e.g., R^(5D)-substituted C₁-C₃ alkyl, R^(5D)-substituted C₁-C₂alkyl, or R^(5D)-substituted methyl) or R^(5D)-substituted 2 to 8membered heteroalkyl (e.g., R^(5D)-substituted 2 to 6 memberedheteroalkyl, R^(5D)-substituted 2 to 5 membered heteroalkyl, orR^(5D)-substituted 2 to 4 membered heteroalkyl). In embodiments, R^(5D)is independently halogen, oxo, —CX^(5D) ₃, —CHX^(5D) ₂, —CH₂X^(5D),—OCX^(5D) ₃, —OCH₂X^(5D), —OCHX^(5D) ₂, —CN, —OH, —SH, —NH₂, —COOH,—CONH₂, —NO₂, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂,—NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —N₃, —SF₅, —NH₃ ⁺,—SO₃—, —OPO₃H⁻, —SCN, or —ONO₂. In embodiments, R^(5B) is independentlyhydrogen, halogen, —CX^(5B) ₃, —CHX^(5B) ₂, —CH₂X^(5B), —OCX^(5B) ₃,—OCH₂X^(5B), —OCHX^(5B) ₂, —CN, —OH, —SH, —NH₂, —COOH, —CONH₂, —NO₂,—SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H,—NHC(O)H, —NHC(O)OH, —NHOH, —N₃, —SF₅, —NH₃ ⁺, —SO₃ ⁻, —OPO₃H⁻, —SCN,—ONO₂, R^(5F)-substituted C₁-C₄ alkyl, (e.g., R^(5F)-substituted C₁-C₃alkyl, R^(5F)-substituted C₁-C₂ alkyl, or R^(5F)-substituted methyl) orR^(5F)-substituted 2 to 8 membered heteroalkyl (e.g., R^(5F)-substituted2 to 6 membered heteroalkyl, R^(5F)-substituted 2 to 5 memberedheteroalkyl, or R^(5F)-substituted 2 to 4 membered heteroalkyl). Inembodiments, R^(5F) is independently halogen, oxo, —CX^(5F) ₃, —CHX^(5F)₂, —CH₂X^(5F), —OCX^(5F) ₃, —OCH₂X^(5F), —OCHX^(5F) ₂, —CN, —OH, —SH,—NH₂, —COOH, —CONH₂, —NO₂, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —N₃,—SF₅, —NH₃ ⁺, —SO₃ ⁻, —OPO₃H, —SCN, or —ONO₂. In embodiments, R^(5A) andR^(5B) are combined to form an oxo. The symbols X^(5A), X^(5B), X^(5D),and X^(5F) are independently —F, —Cl, —Br, or —I.

In embodiments, the -polymerase-compatible cleavable moiety is:

In embodiments, the -polymerase-compatible cleavable moiety is:

In embodiments, R⁷ is substituted or unsubstituted cycloalkyl (e.g.,C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆), substituted or unsubstitutedheterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6membered, 4 to 5 membered, or 5 to 6 membered), substituted orunsubstituted aryl (e.g., C₆-C₁₀ or phenyl), or substituted orunsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered, or 5to 6 membered).

In embodiments, R⁷ is substituted or unsubstituted cycloalkyl (e.g.,C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆). In embodiments, R⁷ is substitutedcycloalkyl (e.g., C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆). In embodiments, R⁷ isan unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆). Inembodiments, R⁷ is substituted or unsubstituted heterocycloalkyl (e.g.,3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5to 6 membered). In embodiments, R⁷ is substituted heterocycloalkyl(e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5membered, or 5 to 6 membered). In embodiments, R⁷ is an unsubstitutedheterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6membered, 4 to 5 membered, or 5 to 6 membered).

In embodiments, R⁷ is substituted or unsubstituted aryl (e.g., C₆-C₁₀,C₁₀, or phenyl). In embodiments, R⁷ is substituted aryl (e.g., C₆-C₁₀,C₁₀, or phenyl). In embodiments, R⁷ is unsubstituted aryl (e.g., C₆-C₁₀,C₁₀, or phenyl). In embodiments, R⁷ is unsubstituted phenyl. Inembodiments, R⁷ is substituted or unsubstituted heteroaryl (e.g., 5 to10, 5 to 9, or 5 to 6 membered). In embodiments, R⁷ is substitutedheteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered). In embodiments,R⁷ is unsubstituted heteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6membered). In embodiments, R⁷ is a substituted or unsubstituted 5membered heteroaryl. In embodiments, R⁷ is a substituted orunsubstituted 6 membered heteroaryl. In embodiments, R⁷ is a substitutedor unsubstituted 7 membered heteroaryl. In embodiments, R⁷ is anunsubstituted 5 membered heteroaryl. In embodiments, R⁷ is anunsubstituted 6 membered heteroaryl. In embodiments, R⁷ is anunsubstituted 7 membered heteroaryl.

In embodiments, R⁷ is R^(7A)-substituted or unsubstituted aryl (e.g.,C₆-C₁₀, C₁₀, or phenyl), or R^(7A)-substituted or unsubstitutedheteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered). R^(7A) isindependently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂, —CHBr₂,—CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂, —COOH,—CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂,—NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃, —OCF₃, —OCBr₃,—OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl, —OCH₂Br, —OCH₂I,—OCH₂F, —N₃, —SF₅, —OPO₃H, R^(7B)-substituted or unsubstituted alkyl(e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄), R^(7B)-substituted orunsubstituted heteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to6, or 2 to 4 membered), R^(7B)-substituted or unsubstituted cycloalkyl(e.g., C₃-C₈, C₃-C₆, or C₅-C₆), R^(7B)-substituted or unsubstitutedheterocycloalkyl (e.g., 3 to 8, 3 to 6, or 5 to 6 membered),R^(7B)-substituted or unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, or phenyl),or R^(7B)-substituted or unsubstituted heteroaryl (e.g., 5 to 10, 5 to9, or 5 to 6 membered). In embodiments, R^(7A) is independently,halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂, —CHBr₂, —CHF₂, —CHI₂, —CH₂Cl,—CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H,—SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H,—NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃, —OCF₃, —OCBr₃, —OCI₃, —OCHCl₂,—OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl, —OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, or—OPO₃H. R^(7B) is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃,—CHCl₂, —CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, R^(7C)-substituted or unsubstitutedalkyl (e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄),R^(7C)-substituted or unsubstituted heteroalkyl (e.g., 2 to 20, 8 to 20,2 to 10, 2 to 8, 2 to 6, or 2 to 4 membered), R^(7C)-substituted orunsubstituted cycloalkyl (e.g., C₃—C₈, C₃-C₆, or C₅-C₆),R^(7C)-substituted or unsubstituted heterocycloalkyl (e.g., 3 to 8, 3 to6, or 5 to 6 membered), R^(7C)-substituted or unsubstituted aryl (e.g.,C₆-C₁₀, C₁₀, or phenyl), or R^(7C)-substituted or unsubstitutedheteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered). R^(7C) isindependently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂, —CHBr₂,—CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂, —COOH,—CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂,—NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃, —OCF₃, —OCBr₃,—OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl, —OCH₂Br, —OCH₂I,—OCH₂F, —N₃, —SF₅, unsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₈,C₁-C₆, or C₁-C₄), unsubstituted heteroalkyl (e.g., 2 to 20, 8 to 20, 2to 10, 2 to 8, 2 to 6, or 2 to 4 membered), unsubstituted cycloalkyl(e.g., C₃-C₈, C₃-C₆, or C₅-C₆), unsubstituted heterocycloalkyl (e.g., 3to 8, 3 to 6, or 5 to 6 membered), unsubstituted aryl (e.g., C₆-C₁₀,C₁₀, or phenyl), or unsubstituted heteroaryl (e.g., 5 to 10, 5 to 9, or5 to 6 membered).

In embodiments, R⁷ is R^(7A)-substituted cycloalkyl (e.g., C₃-C₈, C₃-C₆,C₄-C₆, or C₅-C₆). In embodiments, R⁷ is R^(7A)-substitutedheterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6membered, 4 to 5 membered, or 5 to 6 membered). In embodiments, R^(7A)is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂, —CHBr₂,—CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂, —COOH,—CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂,—NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃, —OCF₃, —OCBr₃,—OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl, —OCH₂Br, —OCH₂I,—OCH₂F, —N₃, —SF₅, —OPO₃H, R^(7B)-substituted or unsubstituted alkyl(e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄), R^(7B)-substituted orunsubstituted heteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to6, or 2 to 4 membered), R^(7B)-substituted or unsubstituted cycloalkyl(e.g., C₃-C₈, C₃-C₆, or C₅-C₆), R^(7B)-substituted or unsubstitutedheterocycloalkyl (e.g., 3 to 8, 3 to 6, or 5 to 6 membered),R^(7B)-substituted or unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, or phenyl),or R^(7B)-substituted or unsubstituted heteroaryl (e.g., 5 to 10, 5 to9, or 5 to 6 membered).

Two adjacent R^(7A) substituents may optionally be joined to form asubstituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. Two adjacent R^(7A) substituents mayoptionally be joined to form a R^(7B)-substituted or unsubstitutedcycloalkyl, R^(7B)-substituted or unsubstituted heterocycloalkyl,R^(7B)-substituted or unsubstituted aryl, or R^(7B)-substituted orunsubstituted heteroaryl. Two adjacent R^(7A) substituents mayoptionally be joined to form a R^(7B)-substituted cycloalkyl,R^(7B)-substituted heterocycloalkyl, R^(7B)-substituted aryl, orR^(7B)-substituted heteroaryl. Two adjacent R^(7A) substituents mayoptionally be joined to form an unsubstituted cycloalkyl, unsubstitutedheterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl.

In embodiments, R⁷ is

wherein R^(7A) is as described herein and z7 is an integer from 0 to 5.In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R^(7A) is —CN, —NO₂, —CF₃, —N₃, —NH₂, —OMe, —OH, —F,—Cl, or —CH₃. In embodiments, R^(7A) is —N₃, —NH₂, —OMe, —OH, —F, —Cl,or —CH₃. In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

wherein z7 and R^(7B) is as described herein. In embodiments, z7 is 4.In embodiments, z7 is 3. In embodiments, z7 is 2. In embodiments, z7is 1. In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

wherein R^(7B) is as described herein.

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is a moiety depicted in Table 4.

In embodiments, R⁷ is

In embodiments R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In

embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, z7 is an integer from 0 to 2. In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments R7 is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments R⁷ is

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In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

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In embodiments, R⁷ is

In embodiments, R⁷ is

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In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁸ is substituted alkyl. In embodiments, R⁸ issubstituted C₁-C₆ or C₁-C₄ alkyl. In embodiments, R⁸ isR^(8A)-substituted C₁-C₆ or C₁-C₄ alkyl. R^(8A) is independently oxo,halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂, —CHBr₂, —CHF₂, —CHI₂, —CH₂Cl,—CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H,—SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H,—NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃, —OCF₃, —OCBr₃, —OCI₃, —OCHCl₂,—OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl, —OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅,unsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄),unsubstituted heteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to6, or 2 to 4 membered), unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, orC₅-C₆), unsubstituted heterocycloalkyl (e.g., 3 to 8, 3 to 6, or 5 to 6membered), unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, or phenyl), orunsubstituted heteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered).

In embodiments, R⁸ is unsubstituted C₁-C₆ or C₁-C₄ alkyl. Inembodiments, R⁸ is unsubstituted C₁-C₄ alkyl. In embodiments, R⁸ isunsubstituted C₁-C₆ alkyl. In embodiments, R′ is unsubstituted methyl.In embodiments, R⁸ is unsubstituted C₂ alkyl. In embodiments, R⁸ isunsubstituted C₃ alkyl. In embodiments, R⁸ is unsubstituted C₄ alkyl. Inembodiments, R⁸ is unsubstituted C₅ alkyl. In embodiments, R⁸ isunsubstituted C₆ alkyl.

In embodiments, R⁸ is unsubstituted C₁-C₆ or C₁-C₄ saturated alkyl. Inembodiments, R′ is unsubstituted C₁-C₄ saturated alkyl. In embodiments,R⁸ is unsubstituted C₁-C₆ saturated alkyl. In embodiments, R⁸ isunsubstituted methyl. In embodiments, R⁸ is unsubstituted C₂ saturatedalkyl. In embodiments, R⁸ is unsubstituted C₃ saturated alkyl. Inembodiments, R⁸ is unsubstituted C₄ saturated alkyl. In embodiments, R⁸is unsubstituted C₅ saturated alkyl. In embodiments, R⁸ is unsubstitutedC₆ saturated alkyl.

In embodiments, B¹ is a cytosine or a derivative thereof, guanine or aderivative thereof, adenine or a derivative thereof, thymine or aderivative thereof, uracil or a derivative thereof, hypoxanthine or aderivative thereof, xanthine or a derivative thereof, 7-methylguanine ora derivative thereof, 5,6-dihydrouracil or a derivative thereof,5-methylcytosine or a derivative thereof, or 5-hydroxymethylcytosine ora derivative thereof.

In embodiments, B¹ is a monovalent nucleobase, or a derivative thereof.In embodiments, B¹ is a monovalent cytosine or a derivative thereof,monovalent guanine or a derivative thereof, monovalent adenine or aderivative thereof, monovalent thymine or a derivative thereof,monovalent uracil or a derivative thereof, monovalent hypoxanthine or aderivative thereof, monovalent xanthine or a derivative thereof,monovalent 7-methylguanine or a derivative thereof, monovalent5,6-dihydrouracil or a derivative thereof, monovalent 5-methylcytosineor a derivative thereof, or monovalent 5-hydroxymethylcytosine or aderivative thereof. In embodiments, B¹ is a monovalent cytosine or aderivative thereof. In embodiments, B¹ is a monovalent guanine or aderivative thereof. In embodiments, B¹ is a monovalent adenine or aderivative thereof. In embodiments, B¹ is a monovalent thymine or aderivative thereof. In embodiments, B¹ is a monovalent uracil or aderivative thereof. In embodiments, B¹ is a monovalent hypoxanthine or aderivative thereof. In embodiments, B¹ is a monovalent xanthine or aderivative thereof. In embodiments, B¹ is a monovalent 7-methylguanineor a derivative thereof. In embodiments, B¹ is a monovalent5,6-dihydrouracil or a derivative thereof. In embodiments, B¹ is amonovalent 5-methylcytosine or a derivative thereof. In embodiments, B¹is a monovalent 5-hydroxymethylcytosine or a derivative thereof. Inembodiments, B¹ is a monovalent cytosine. In embodiments, B¹ is amonovalent guanine. In embodiments, B¹ is a monovalent adenine. Inembodiments, B¹ is a monovalent thymine. In embodiments, B¹ is amonovalent uracil. In embodiments, B¹ is a monovalent hypoxanthine. Inembodiments, B¹ is a monovalent xanthine. In embodiments, B¹ is amonovalent 7-methylguanine. In embodiments, B¹ is a monovalent5,6-dihydrouracil. In embodiments, B¹ is a monovalent 5-methylcytosine.In embodiments, B¹ is a monovalent 5-hydroxymethylcytosine.

In embodiments, B¹ is

In embodiments, B¹ is

In embodiments, B¹ is

In embodiments, B¹ is

In embodiments, B¹ is

In embodiments, B¹ is

In embodiments, B¹ is

In embodiments, B¹ is

In embodiments, B¹ is

In embodiments, B¹ is

In embodiments, B¹ is

In embodiments, B¹ is

In embodiments, B¹ is

In embodiments, B¹ is

In embodiments, B¹ is

In embodiments, B¹ is

In embodiments, B¹ is

In embodiments, B¹ is

In embodiments, B¹ is

In embodiments, B¹ is

In embodiments, B¹ is

In embodiments, B¹ includes a substituted or unsubstituted propargylamine moiety, which may further include S—S linker, fluorophores orprotecting group. In embodiments, the propargyl amine moiety may furtherinclude at least one or more fluorophores. In embodiments, the propargylamine moiety may further be linked via a linker (e.g., an S—S linker) toat least one or more fluorophores. In embodiments, the propargyl aminemoiety may further include at least one or more protecting groups. Inembodiments, the propargyl amine moiety may further be linked to a S—S—linker, which may be connected to at least one or more protectinggroups.

In embodiments, B¹ is a divalent nucleobase. In embodiments, B¹ is

In embodiments, B¹ is

In embodiments, B is a divalent nucleobase. In embodiments, B is

In embodiments, B is

In embodiments, B¹ is —B-L¹⁰⁰-R⁴. B is a divalent cytosine or aderivative thereof, divalent guanine or a derivative thereof, divalentadenine or a derivative thereof, divalent thymine or a derivativethereof, divalent uracil or a derivative thereof, divalent hypoxanthineor a derivative thereof, divalent xanthine or a derivative thereof,divalent 7-methylguanine or a derivative thereof, divalent5,6-dihydrouracil or a derivative thereof, divalent 5-methylcytosine ora derivative thereof, or divalent 5-hydroxymethylcytosine or aderivative thereof. L¹⁰⁰ is a divalent linker; and R⁴ is a detectablemoiety. In embodiments, L¹⁰⁰ includes a thio-trigger moiety.

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, B is a divalent cytosine or a derivative thereof,divalent guanine or a derivative thereof, divalent adenine or aderivative thereof, divalent thymine or a derivative thereof, divalenturacil or a derivative thereof, divalent hypoxanthine or a derivativethereof, divalent xanthine or a derivative thereof, divalent7-methylguanine or a derivative thereof, divalent 5,6-dihydrouracil or aderivative thereof, divalent 5-methylcytosine or a derivative thereof,or divalent 5-hydroxymethylcytosine or a derivative thereof. Inembodiments, B is a divalent cytosine or a derivative thereof. Inembodiments, B is a divalent guanine or a derivative thereof. Inembodiments, B is a divalent adenine or a derivative thereof. Inembodiments, B is a divalent thymine or a derivative thereof. Inembodiments, B is a divalent uracil or a derivative thereof. Inembodiments, B is a divalent hypoxanthine or a derivative thereof. Inembodiments, B is a divalent xanthine or a derivative thereof. Inembodiments, B is a divalent 7-methylguanine or a derivative thereof. Inembodiments, B is a divalent 5,6-dihydrouracil or a derivative thereof.In embodiments, B is a divalent 5-methylcytosine or a derivativethereof. In embodiments, B is a divalent 5-hydroxymethylcytosine or aderivative thereof. In embodiments, B is a divalent cytosine. Inembodiments, B is a divalent guanine. In embodiments, B is a divalentadenine. In embodiments, B is a divalent thymine. In embodiments, B is adivalent uracil. In embodiments, B is a divalent hypoxanthine. Inembodiments, B is a divalent xanthine. In embodiments, B is a divalent7-methylguanine. In embodiments, B is a divalent 5,6-dihydrouracil. Inembodiments, B is a divalent 5-methylcytosine. In embodiments, B is adivalent 5-hydroxymethylcytosine.

In embodiments, L¹⁰⁰ is -L¹⁰¹-L¹⁰²-L¹⁰³-L¹⁰⁴-L¹⁰⁵-. In embodiments, L¹⁰⁰is independently a bioconjugate linker; a cleavable linker, aself-immolative linker, a linker capable of dendritic amplification ofsignal (e.g., capable of increasing fluorescence by releasingfluorophores from the remainder of the linker, optionally wherein thefluorescence is increased following release), a trivalent linker, or aself-immolative dendrimer linker (e.g., capable of increasingfluorescence by releasing fluorophores from the remainder of thelinker). In embodiments, L¹⁰⁰ is independently a bioconjugate linker. Inembodiments, L¹⁰⁰ is independently a cleavable linker. In embodiments,L¹⁰⁰ is independently a self-immolative linker. In embodiments, L¹⁰⁰ isindependently a linker capable of dendritic amplification of signal(e.g., capable of increasing fluorescence by releasing fluorophores). Inembodiments, L¹⁰⁰ is independently a trivalent linker. In embodiments,L¹⁰⁰ is independently a self-immolative dendrimer linker (e.g., capableof increasing fluorescence by releasing fluorophores).

In embodiments, L¹⁰⁰ includes

wherein R⁹ is independently hydrogen, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃,—CHCl₂, —CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl. Inembodiments, R⁹ is substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl. Inembodiments, R⁹ is a moiety represented by R⁷. In embodiments, L¹⁰⁰includes

or w wherein R⁹ is as described herein. In embodiments, L¹⁰⁰ includes

wherein R⁹ is as described herein.

In embodiments, L¹⁰⁰ includes a thio-trigger moiety. In embodiments,L¹⁰⁰ includes

wherein R¹⁰² is as described herein. In embodiments, L¹⁰⁰ includes

wherein R¹⁰² is as described herein. In embodiments, L¹⁰⁰ includes

wherein R¹⁰² is as described herein. In embodiments, R¹⁰² isunsubstituted C₁-C₄ alkyl. In embodiments, R¹⁰² is unsubstituted C₁alkyl. In embodiments, R¹⁰² is unsubstituted C₂ alkyl. In embodiments,R¹⁰² is unsubstituted C₃ alkyl. In embodiments, R¹⁰² is unsubstituted C₄alkyl. In embodiments, L¹⁰⁰ includes

wherein R¹⁰² is as described herein. In embodiments, L¹⁰⁰ includes

wherein R¹⁰² is as described herein.

In embodiments, L¹⁰⁰ includes

wherein R¹⁰² is as described herein. In embodiments, R¹⁰² isunsubstituted C₁-C₄ alkyl. In embodiments, R¹⁰² is unsubstituted Cialkyl. In embodiments, R¹⁰² is unsubstituted C₂ alkyl. In embodiments,R¹⁰² is unsubstituted C₃ alkyl. In embodiments, R¹⁰² is unsubstituted C₄alkyl.

L¹⁰¹, L¹⁰², L¹⁰³, L¹⁰⁴, and L¹⁰⁵ are independently a bond, —NH—, —S—,—O—, —C(O)—, —C(O)O—, —OC(O)—, —NHC(O)—, —C(O)NH—, —NHC(O)NH—,—NHC(NH)NH—, —C(S)—, —N═N—, —SS—, thio-trigger moiety, substituted orunsubstituted alkylene (e.g., —CH(OH)— or —C(CH₂)—), substituted orunsubstituted heteroalkylene, substituted or unsubstitutedcycloalkylene, substituted or unsubstituted heterocycloalkylene,substituted or unsubstituted arylene, or substituted or unsubstitutedheteroarylene; a bioconjugate linker; a cleavable linker, aself-immolative linker, a linker capable of dendritic amplification ofsignal (e.g., capable of increasing fluorescence by releasingfluorophores from the remainder of the linker), a trivalent linker, or aself-immolative dendrimer linker (e.g., capable of increasingfluorescence by releasing fluorophores from the remainder of thelinker). In embodiments, L¹⁰¹, L¹⁰², L¹⁰³, L¹⁰⁴, and L¹⁰⁵ areindependently a bond, —NH—, —S—, —O—, —C(O)—, —C(O)O—, —OC(O)—,—NHC(O)—, —C(O)NH—, —NHC(O)NH—, —NHC(NH)NH—, —C(S)—, —N═N—, —SS—,substituted or unsubstituted alkylene (e.g., —CH(OH)— or —C(CH₂)—),substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene; a bioconjugate linker; or acleavable linker. In embodiments, L¹⁰¹, L¹⁰², L¹⁰³, L¹⁰⁴, and L¹⁰⁵independently includes PEG. In embodiments, L¹⁰¹, L¹⁰², L¹⁰³, L¹⁰⁴, andL¹⁰⁵ independently includes

wherein z100 is independently 1 to 8. In embodiments, z100 is 1. Inembodiments, z100 is 2. In embodiments, z100 is 3. In embodiments, z100is 4. In embodiments, z100 is 5. In embodiments, z100 is 6. Inembodiments, z100 is 7. In embodiments, z100 is 8. In embodiments, z100is 2 to 8. In embodiments, z100 is 4 to 6.

In embodiments, at least one of L¹⁰¹, L¹⁰², L¹⁰³, L¹⁰⁴, and L¹⁰⁵independently includes

wherein R⁹ is as described herein. In embodiments, at least one of L¹⁰¹,L¹⁰², L¹⁰³, L¹⁰⁴, and L¹⁰⁵ independently includes

wherein R⁹ is as described herein. In embodiments, at least one of L¹⁰¹,L¹⁰², L¹⁰³, L¹⁰⁴, and L¹⁰⁵ independently includes

wherein R⁹ is as described herein. In embodiments, at least one of L¹⁰¹,L¹⁰², L¹⁰³, L¹⁰⁴, and L¹⁰⁵ independently includes

wherein R¹⁰² is as described herein. In embodiments, at least one ofL¹⁰¹, L¹⁰², L¹⁰³, L¹⁰⁴, and L¹⁰⁵ independently includes

wherein R¹⁰² is as described herein. In embodiments, at least one ofL¹⁰¹, L¹⁰², L¹⁰³, L¹⁰⁴, and L¹⁰⁵ independently includes

wherein R¹⁰² is as described herein. In embodiments, at least one ofL¹⁰¹, L¹⁰², L¹⁰³, L¹⁰⁴, and L¹⁰⁵ independently includes

In embodiments, at least one of L¹⁰¹, L¹⁰², L¹⁰³, L¹⁰⁴, and L¹⁰⁵independently includes

wherein R⁹ is as described herein. In embodiments, at least one of L¹⁰¹,L¹⁰², L¹⁰³, L¹⁰⁴, and L¹⁰⁵ independently includes

wherein R⁹ is as described herein. In embodiments, at least one of L¹⁰¹,L¹⁰², L¹⁰³, L¹⁰⁴ and L¹⁰⁵ independently includes

wherein R⁹ is as described herein. In embodiments, at least one of L¹⁰¹,L¹⁰², L¹⁰³, L¹⁰⁴, and L¹⁰⁵ independently includes

wherein R¹⁰² is as described herein. In embodiments, at least one ofL¹⁰¹, L¹⁰², L¹⁰³, L¹⁰⁴, and L¹⁰⁵ independently includes

wherein R¹⁰² is as described herein. In embodiments, at least one ofL¹⁰¹, L¹⁰², L¹⁰³, L¹⁰⁴, and L¹⁰⁵ independently includes

wherein R¹⁰² is as described herein. In embodiments, at least one ofL¹⁰¹, L¹⁰², L¹⁰³, L¹⁰⁴, and L¹⁰⁵ independently includes

In embodiments, L¹⁰⁰ is -L¹⁰¹-L¹⁰²-L¹⁰³-L¹⁰⁴-L¹⁰⁵-. In embodiments,L¹⁰¹, L¹⁰², L¹⁰³, L¹⁰⁴, and L¹⁰⁵ are independently a bond, —NH—, —O—,—C(O)—, —C(O)NH—, —NHC(O)—, —NHC(O)NH—, —C(O)O—, —OC(O)—, substituted orunsubstituted alkylene, substituted or unsubstituted heteroalkylene,substituted or unsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene.

In embodiments, L¹⁰⁰ is -L¹⁰¹-O—CH(—SR¹⁰⁰)-L¹⁰³-L¹⁰⁴-L¹⁰⁵-;-L¹⁰¹-O—C(CH₃)(—SR¹⁰⁰)-L¹⁰³-L¹⁰⁴-L¹⁰⁵-; -L¹⁰¹-O—CH(N₃)-L¹⁰³-L¹⁰⁴-L¹⁰⁵-;or -L¹⁰¹-O—CH(N₃)—CH₂—O-L¹⁰⁴-L¹⁰⁵-; wherein L¹⁰¹, L¹⁰³, L¹⁰⁴, and L¹⁰⁵are independently a bond, —NH—, —O—, —C(O)—, —C(O)NH—, —NHC(O)—,—NHC(O)NH—, —C(O)O—, —OC(O)—, substituted or unsubstituted alkylene,substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene; R¹⁰⁰ is —SR¹⁰² or —CN; andR¹⁰² is R^(102B)-substituted or unsubstituted C₁-C₄ alkyl. Inembodiments, R¹⁰² is unsubstituted C₁-C₄ alkyl. In embodiments, R¹⁰⁰ is—SO₃H.

In embodiments, L¹⁰⁰ is -L¹⁰¹-O—CH(—SR¹⁰⁰)-L¹⁰³-L¹⁰⁴-L¹⁰⁵- or-L¹⁰¹-O—C(CH₃)(—SR¹⁰⁰)-L¹⁰³-L¹⁰⁴-L¹⁰⁵-; wherein L¹⁰¹ is independently asubstituted or unsubstituted C₁-C₄ alkylene or substituted orunsubstituted 8 to 20 membered heteroalkylene; L¹⁰³ is independently abond or substituted or unsubstituted 2 to 10 membered heteroalkylene;L¹⁰⁴ is independently a bond, substituted or unsubstituted 4 to 18membered heteroalkylene, or substituted or unsubstituted phenylene; L¹⁰⁵is independently bond or substituted or unsubstituted 4 to 18 memberedheteroalkylene; R¹⁰⁰ is —SR¹⁰² or —CN; and R¹⁰² is R^(102B)-substitutedor unsubstituted C₁-C₄ alkyl. In embodiments, R¹⁰² is unsubstitutedC₁-C₄ alkyl. In embodiments, R¹⁰⁰ is —SO₃H.

In embodiments, L¹⁰⁰ is -L¹⁰¹-O—CH(—SR¹⁰⁰)-L¹⁰³-L¹⁰⁴-L¹⁰⁵- or-L¹⁰¹-O—C(CH₃)(—SR¹⁰⁰)-L¹⁰³-L¹⁰⁴-L¹⁰⁵-; wherein L¹⁰¹, L¹⁰³, and L¹⁰⁵ areindependently a bond, —NH—, —O—, —C(O)—, —C(O)NH—, —NHC(O)—, —NHC(O)NH—,—C(O)O—, —OC(O)—, substituted or unsubstituted alkylene, substituted orunsubstituted heteroalkylene, substituted or unsubstitutedcycloalkylene, substituted or unsubstituted heterocycloalkylene,substituted or unsubstituted arylene, or substituted or unsubstitutedheteroarylene; L¹⁰⁴ is unsubstituted phenylene; R¹⁰⁰ is —SR¹⁰² or —CN;and R¹⁰² is R^(102B)-substituted or unsubstituted C₁-C₄ alkyl. Inembodiments, R¹⁰² is unsubstituted C₁-C₄ alkyl. In embodiments, R¹⁰⁰ is—SO₃H.

In embodiments, L¹⁰¹, L¹⁰², L¹⁰³, L¹⁰⁴, and L¹⁰⁵ are independently abond, —NH—, —S—, —O—, —C(O)—, —C(O)O—, —OC(O)—, —NHC(O)—, —C(O)NH—,—NHC(O)NH—, —NHC(NH)NH—, —C(S)—, substituted or unsubstituted alkylene(e.g., —CH(OH)— or —C(CH₂)—), substituted or unsubstitutedheteroalkylene, substituted or unsubstituted cycloalkylene, substitutedor unsubstituted heterocycloalkylene, substituted or unsubstitutedarylene, or substituted or unsubstituted heteroarylene; a bioconjugatelinker; or a cleavable linker.

In embodiments, L¹⁰¹, L¹⁰², L¹⁰³, L¹⁰⁴, and L¹⁰⁵ are independently abond, —NH—, —S—, —O—, —C(O)—, —C(O)O—, —OC(O)—, —NHC(O)—, —C(O)NH—,—NHC(O)NH—, —NHC(NH)NH—, —C(S)—, substituted or unsubstituted alkylene(e.g., —CH(OH)— or —C(CH₂)—), substituted or unsubstitutedheteroalkylene, substituted or unsubstituted cycloalkylene, substitutedor unsubstituted heterocycloalkylene, substituted or unsubstitutedarylene, or substituted or unsubstituted heteroarylene. In embodiments,L¹⁰¹, L¹⁰², L¹⁰³, L¹⁰⁴, or L¹⁰⁵ is independently a bioconjugate linker.In embodiments, L¹⁰¹, L¹⁰², L¹⁰³, L¹⁰⁴, or L¹⁰⁵ is independently acleavable linker. In embodiments, L¹⁰¹, L¹⁰², L¹⁰³, L¹⁰⁴, or L¹⁰⁵ isindependently a self-immolative linker. In embodiments, L¹⁰¹, L¹⁰²,L¹⁰³, L¹⁰⁴, or L¹⁰⁵ is independently a linker capable of dendriticamplification of signal (e.g., capable of increasing fluorescence byreleasing fluorophores). In embodiments, L¹⁰¹, L¹⁰², L¹⁰³, L¹⁰⁴, or L¹⁰⁵is independently a trivalent linker. In embodiments, L¹⁰¹, L¹⁰², L¹⁰³,L¹⁰⁴, or L¹⁰⁵ is independently a self-immolative dendrimer linker (e.g.,capable of increasing fluorescence by releasing fluorophores).

In embodiments, L¹⁰¹, L¹⁰², L¹⁰³, L¹⁰⁴, and L¹⁰⁵ are independently abond, —NH—, —O—, —C(O)—, —C(O)NH—, —NHC(O)—, —NHC(O)NH—, —C(O)O—,—OC(O)—, substituted or unsubstituted alkylene (e.g., —CH(OH)— or—C(CH₂)—), substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene.

In embodiments, L¹⁰¹, L¹⁰², L¹⁰³, L¹⁰⁴, and/or L¹⁰⁵ are independently abond, —NH—, —S—, —O—, —C(O)—, —C(O)O—, —OC(O)—, —CH(OH)—, —NHC(O)—,—C(O)NH—, —NHC(O)NH—, —NHC(NH)NH—, —C(S)—, or —C(CH₂)—.

In embodiments, L¹⁰⁰ is -L¹⁰¹-O—CH(—SR¹⁰⁰)-L¹⁰³-L¹⁰⁴-L¹⁰⁵- or-L¹⁰¹-O—C(CH₃)(—SR¹⁰⁰)-L¹⁰³-L¹⁰⁴-L¹⁰⁵. R¹⁰⁰ is —SR¹⁰² or —CN. Inembodiments, R¹⁰⁰ is —SR¹⁰². In embodiments, R¹⁰⁰ is —CN. R¹⁰² isindependently hydrogen, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂,—CHBr₂, —CHF₂, —CHI₂, —CH₂C₁, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl. Inembodiments, R¹⁰⁰ is —SO₃H.

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

wherein R⁹ is substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl. Inembodiments, R⁹ is substituted or unsubstituted alkyl. In embodiments,R⁹ is substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl. In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

wherein R¹⁰² is as described herein. In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

wherein R¹⁰² is as described herein. In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

Methods for cleaving the disulfide bond of —S—SO₃H bonds are known inthe art, see for example Meguro et al. Tetrahedron Letters 61 (2020)152198, which is incorporated herein by reference in its entirety. Inembodiments, the cleaving agent is aqueous sodium sulfide (Na₂S). Inembodiments, the cleaving agent is TCEP or THPP.

In embodiments, -L¹⁰¹-L¹⁰²-L¹⁰³-L¹⁰⁴-L¹⁰⁵- has the formula:

wherein L¹⁰¹, R¹⁰², and L¹⁰⁵ are as described herein. In embodiments,R¹⁰² is unsubstituted C₁-C₆ alkyl. In embodiments, R¹⁰² is unsubstitutedaryl. In embodiments, R¹⁰² is unsubstituted heteroaryl. In embodiments,-L¹⁰¹-L¹⁰²-L¹⁰³-L¹⁰⁴-L¹⁰⁵- has the formula:

wherein L¹⁰¹, R¹⁰², and L¹⁰⁵ are as described herein. In embodiments,R¹⁰² is unsubstituted C₁-C₆ alkyl. In embodiments, R¹⁰² is unsubstitutedaryl. In embodiments, R¹⁰² is unsubstituted heteroaryl.

In embodiments, L¹⁰⁰ is

wherein L¹⁰¹, L¹⁰³, L¹⁰⁴, L¹⁰⁵, and R⁹ are as described herein. Inembodiments, L¹⁰⁰ is

wherein L¹⁰¹, L¹⁰², L¹⁰⁴, L¹⁰⁵, and R⁹ are as described herein. Inembodiments, L¹⁰⁰ is

wherein L¹⁰¹, L¹⁰², L¹⁰³, L¹⁰⁵, and R⁹ are as described herein. Inembodiments, L¹⁰⁰ is

wherein L¹⁰¹, L¹⁰³, L¹⁰⁴, L¹⁰⁵, and R⁹ are as described herein. Inembodiments, L¹⁰⁰ is

wherein L¹⁰¹, L¹⁰², L¹⁰⁴, L¹⁰⁵, and R⁹ are as described herein. Inembodiments, L¹⁰⁰ is

wherein L¹⁰¹, L¹⁰², L¹⁰³, L¹⁰⁵, and R⁹ are as described herein.

In embodiments, L¹⁰² is

wherein R⁹ is as described herein. In embodiments, L¹⁰³ is

wherein R⁹ is as described herein. In embodiments, L¹⁰⁴ is

wherein R⁹ is as described herein. In embodiments, L¹⁰² is

wherein R⁹ is as described herein. In embodiments, L¹⁰³ is

wherein R⁹ is as described herein. In embodiments, L¹⁰⁴ is

wherein R⁹ is as described herein. In embodiments, L¹⁰² is

wherein R⁹ is as described herein. In embodiments, L¹⁰³ is

wherein R⁹ is as described herein. In embodiments, L¹⁰⁴ is

wherein R⁹ is as described herein.

In embodiments, L¹⁰² is

wherein R¹⁰² is as described herein. In embodiments, L¹⁰³ is

wherein R¹⁰² is as described herein. In embodiments, L¹⁰⁴ is

wherein R¹⁰² is as described herein. In embodiments, L¹⁰² is

wherein R¹⁰² is as described herein. In embodiments, L¹⁰³ is

wherein R¹⁰² is as described herein. In embodiments, L¹⁰⁴ is

wherein R¹⁰² is as described herein. In embodiments, L¹⁰² is

wherein R¹⁰² is as described herein. In embodiments, L¹⁰³ is

wherein R¹⁰² is as described herein. In embodiments, L¹⁰⁴ is

wherein R¹⁰² is as described herein.

In embodiments, R⁹ is independently hydrogen, halogen, —CCl₃, —CBr₃,—CF₃, —CI₃, —CHCl₂, —CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I,—CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCCl₃, —OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, substituted or unsubstituted alkyl(e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄), substituted orunsubstituted heteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to6, or 2 to 4 membered), substituted or unsubstituted cycloalkyl (e.g.,C₃-C₈, C₃-C₆, or C₅-C₆), substituted or unsubstituted heterocycloalkyl(e.g., 3 to 8, 3 to 6, or 5 to 6 membered), substituted or unsubstitutedaryl (e.g., C₆-C₁₀, C₁₀, or phenyl), or substituted or unsubstitutedheteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered).

In embodiments, R⁹ is independently hydrogen, halogen, —CCl₃, —CBr₃,—CF₃, —CI₃, —CHCl₂, —CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I,—CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCCl₃, —OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, R¹⁰-substituted or unsubstitutedalkyl (e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄), R¹⁰-substitutedor unsubstituted heteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2to 6, or 2 to 4 membered), R¹⁰-substituted or unsubstituted cycloalkyl(e.g., C₃-C₈, C₃-C₆, or C₅-C₆), R¹⁰-substituted or unsubstitutedheterocycloalkyl (e.g., 3 to 8, 3 to 6, or 5 to 6 membered),R¹⁰-substituted or unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, or phenyl), orR¹⁰-substituted or unsubstituted heteroaryl (e.g., 5 to 10, 5 to 9, or 5to 6 membered). In embodiments, R⁹ is R¹⁰-substituted or unsubstitutedalkyl (e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄), R¹⁰-substitutedor unsubstituted heteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2to 6, or 2 to 4 membered), R¹⁰-substituted or unsubstituted cycloalkyl(e.g., C₃-C₈, C₃-C₆, or C₅-C₆), R¹⁰-substituted or unsubstitutedheterocycloalkyl (e.g., 3 to 8, 3 to 6, or 5 to 6 membered),R¹⁰-substituted or unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, or phenyl), orR¹⁰-substituted or unsubstituted heteroaryl (e.g., 5 to 10, 5 to 9, or 5to 6 membered). In embodiments, R⁹ is unsubstituted alkyl (e.g., C₁-C₂₀,C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄), unsubstituted heteroalkyl (e.g., 2 to20, 8 to 20, 2 to 10, 2 to 8, 2 to 6, or 2 to 4 membered), unsubstitutedcycloalkyl (e.g., C₃-C₈, C₃-C₆, or C₅-C₆), unsubstitutedheterocycloalkyl (e.g., 3 to 8, 3 to 6, or 5 to 6 membered),unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, or phenyl), or unsubstitutedheteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered).

In embodiments, R⁹ is independently unsubstituted alkyl (e.g., C₁-C₂₀,C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄). In embodiments, R⁹ is independentlyunsubstituted C₁-C₆ alkyl. In embodiments, R⁹ is independentlyunsubstituted C₁-C₄ alkyl. In embodiments, R⁹ is independentlyunsubstituted methyl. In embodiments, R⁹ is independently unsubstitutedethyl. In embodiments, R⁹ is independently unsubstituted propyl. Inembodiments, R⁹ is independently unsubstituted tert-butyl.

In embodiments, R⁹ is independently unsubstituted C₃-C₈ cycloalkyl. Inembodiments, R⁹ is independently unsubstituted C₃-C₆ cycloalkyl. Inembodiments, R⁹ is independently unsubstituted C₅-C₆ cycloalkyl. Inembodiments, R⁹ is independently unsubstituted 3 to 8 memberedheterocycloalkyl. In embodiments, R⁹ is independently unsubstituted 3 to6 membered heterocycloalkyl. In embodiments, R⁹ is independentlyunsubstituted 5 to 6 membered heterocycloalkyl. In embodiments, R⁹ isindependently unsubstituted phenyl. In embodiments, R⁹ is independentlyunsubstituted 5 to 6 membered heteroaryl. In embodiments, R⁹ isindependently unsubstituted 5 membered heteroaryl. In embodiments, R⁹ isindependently unsubstituted 6 membered heteroaryl.

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R¹⁰ is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CN, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —N₃,unsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄),unsubstituted heteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to6, or 2 to 4 membered), unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, orC₅-C₆), unsubstituted heterocycloalkyl (e.g., 3 to 8, 3 to 6, or 5 to 6membered), unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, or phenyl), orunsubstituted heteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered).

In embodiments, L¹⁰⁰ is -L¹⁰¹-O—CH(N₃)-L¹⁰³-L¹⁰⁴-L¹⁰⁵-; and L¹⁰¹, L¹⁰³,L¹⁰⁴, and L¹⁰⁵ are independently a bond, —NH—, —O—, —C(O)—, —C(O)NH—,—NHC(O)—, —NHC(O)NH—, —C(O)O—, —OC(O)—, substituted or unsubstitutedalkylene, substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene. In embodiments, L¹⁰⁰ is-L¹⁰¹-O—CH(N₃)-L¹⁰³-L¹⁰⁴-L¹⁰⁵-; wherein L¹⁰¹ is independently asubstituted or unsubstituted C₁-C₄ alkylene or substituted orunsubstituted 8 to 20 membered heteroalkylene; L¹⁰³ is independently abond or substituted or unsubstituted 2 to 10 membered heteroalkylene;L¹⁰⁴ is independently a bond, substituted or unsubstituted 4 to 18membered heteroalkylene, or substituted or unsubstituted phenylene; andL¹⁰⁵ is independently bond or substituted or unsubstituted 4 to 18membered heteroalkylene. In embodiments, L¹⁰⁰ is-L¹⁰¹-O—CH(N₃)—CH₂—O-L¹⁰⁴-L¹⁰⁵-; wherein L¹⁰¹ and L¹⁰⁵ are independentlya bond, —NH—, —O—, —C(O)—, —C(O)NH—, —NHC(O)—, —NHC(O)NH—, —C(O)O—,—OC(O)—, substituted or unsubstituted alkylene, substituted orunsubstituted heteroalkylene, substituted or unsubstitutedcycloalkylene, substituted or unsubstituted heterocycloalkylene,substituted or unsubstituted arylene, or substituted or unsubstitutedheteroarylene; and L¹⁰⁴ is unsubstituted phenylene.

In embodiments, L¹⁰⁰ is -L¹⁰¹-O—CH(—SSR¹⁰²)-L¹⁰³-L¹⁰⁴-L¹⁰⁵- or-L¹⁰¹-O—C(CH₃)(—SSR¹⁰²)-L¹⁰³-L¹⁰⁴-L¹⁰⁵-. In embodiments, L¹⁰⁰ is-L¹⁰¹-O—CH(—SSR¹⁰²)-L¹⁰³-L¹⁰⁴-L¹⁰⁵-. In embodiments, L¹⁰⁰ is-L¹⁰¹-O—C(CH₃)(—SSR¹⁰²)-L¹⁰³-L¹⁰⁴-L¹⁰⁵-. In embodiments, L¹⁰⁰ is-L¹⁰¹-O—CH(—SCN)-L¹⁰³-L¹⁰⁴-L¹⁰⁵- or -L¹⁰¹-O—C(CH₃)(—SCN)-L³-L¹⁰⁴-L¹⁰⁵-.In embodiments, L¹⁰⁰ is -L¹⁰¹-O—CH(—SCN)-L¹⁰³-L¹⁰⁴-L¹⁰⁵-. Inembodiments, L¹⁰⁰ is -L¹⁰¹-O—C(CH₃)(—SCN)-L¹⁰³-L¹⁰⁴-L¹⁰⁵-.

In embodiments, L¹⁰¹, L¹⁰³, L¹⁰⁴, and L¹⁰⁵ are independently a bond,—NH—, —O—, —C(O)—, —C(O)NH—, —NHC(O)—, —NHC(O)NH—, —C(O)O—, —OC(O)—,substituted or unsubstituted alkylene, substituted or unsubstitutedheteroalkylene, substituted or unsubstituted cycloalkylene, substitutedor unsubstituted heterocycloalkylene, substituted or unsubstitutedarylene, or substituted or unsubstituted heteroarylene; and R¹⁰² isunsubstituted C₁-C₄ alkyl.

In embodiments, L¹⁰¹, L¹⁰³, L¹⁰⁴, and L¹⁰⁵ are independently a bond,—NH—, —O—, —C(O)—, —C(O)NH—, —NHC(O)—, —NHC(O)NH—, —C(O)O—, —OC(O)—,substituted or unsubstituted alkylene (e.g., —CH(OH)— or —C(CH₂)—),substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene.

In embodiments, R¹⁰² is unsubstituted C₁-C₄ alkyl. In embodiments, R¹⁰²is unsubstituted methyl. In embodiments, R¹⁰² is unsubstituted ethyl. Inembodiments, R¹⁰² is unsubstituted propyl. In embodiments, R¹⁰² isunsubstituted isopropyl. In embodiments, R¹⁰² is unsubstituted butyl. Inembodiments, R¹⁰² is unsubstituted tert-butyl.

In embodiments, L¹⁰⁰ is -L¹⁰¹-O—CH(—SR¹⁰⁰)-L¹⁰³-L¹⁰⁴-L¹⁰⁵- or-L¹⁰¹-O—C(CH₃)(—SR¹⁰⁰)-L¹⁰³-L¹⁰⁴-L¹⁰⁵-. In embodiments, L¹⁰⁰ is-L¹⁰¹-O—CH(—SR¹⁰⁰)-L¹⁰³-L¹⁰⁴-L¹⁰⁵-. In embodiments, L¹⁰⁰ is-L¹⁰¹-O—C(CH₃)(—SR¹⁰⁰)-L⁰³-L¹⁴-L¹⁰⁵-.

In embodiments, L¹⁰⁰ is -L¹⁰¹-O—CH(—SSR¹⁰²)-L¹⁰³-L¹⁰⁴-L¹⁰⁵- or-L¹⁰¹-O—C(CH₃)(—SSR¹⁰²)-L¹⁰³-L¹⁰⁴-L¹⁰⁵-. In embodiments, L¹⁰⁰ is-L¹⁰¹-O—CH(—SSR¹⁰²)-L¹⁰³-L¹⁰⁴-L¹⁰⁵-. In embodiments, L¹⁰⁰ is-L¹⁰¹-O—C(CH₃)(—SSR¹⁰²)-L¹⁰³-L¹⁰⁴-L¹⁰⁵-.

In embodiments, L¹⁰⁰ is -L¹⁰¹-O—CH(—SCN)-L¹⁰³-L¹⁰⁴-L¹⁰⁵- or-L¹⁰¹-O—C(CH₃)(—SCN)-L¹⁰³-L¹⁰⁴-L¹⁰⁵-. In embodiments, L¹⁰⁰ is-L¹⁰¹-O—CH(—SCN)-L¹⁰³-L¹⁰⁴-L¹⁰⁵-. In embodiments, L¹⁰⁰ is-L¹⁰¹-O—C(CH₃)(—SCN)-L¹⁰³-L¹⁰⁴-L¹⁰⁵-.

In embodiments, L¹⁰¹ is independently a substituted or unsubstitutedC₁-C₄ alkylene or substituted or unsubstituted 8 to 20 memberedheteroalkylene; L¹⁰³ is independently a bond or substituted orunsubstituted 2 to 10 membered heteroalkylene; L¹⁰⁴ is independently abond, substituted or unsubstituted 4 to 18 membered heteroalkylene, orsubstituted or unsubstituted phenylene; L¹⁰⁵ is independently bond orsubstituted or unsubstituted 4 to 18 membered heteroalkylene; and R¹⁰²is unsubstituted C₁-C₄ alkyl. In embodiments, L¹⁰¹ is independently asubstituted or unsubstituted C₁-C₄ alkylene or substituted orunsubstituted 8 to 20 membered heteroalkylene. In embodiments, L¹⁰³ isindependently a bond or substituted or unsubstituted 2 to 10 memberedheteroalkylene. In embodiments, L¹⁰⁴ is independently a bond,substituted or unsubstituted 4 to 18 membered heteroalkylene, orsubstituted or unsubstituted phenylene. In embodiments, L¹⁰⁵ isindependently bond or substituted or unsubstituted 4 to 18 memberedheteroalkylene.

In embodiments, L¹⁰¹, L¹⁰³, and L¹⁰⁵ are independently a bond, —NH—,—O—, —C(O)—, —C(O)NH—, —NHC(O)—, —NHC(O)NH—, —C(O)O—, —OC(O)—,substituted or unsubstituted alkylene (e.g., —CH(OH)— or —C(CH₂)—),substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene; L¹⁰⁴ is unsubstitutedphenylene; and R¹⁰² is unsubstituted C₁-C₄ alkyl. In embodiments, L¹⁰¹,L¹⁰³, and L¹⁰⁵ are independently a bond, —NH—, —O—, —C(O)—, —C(O)NH—,—NHC(O)—, —NHC(O)NH—, —C(O)O—, —OC(O)—, substituted or unsubstitutedalkylene (e.g., —CH(OH)— or —C(CH₂)—), substituted or unsubstitutedheteroalkylene, substituted or unsubstituted cycloalkylene, substitutedor unsubstituted heterocycloalkylene, substituted or unsubstitutedarylene, or substituted or unsubstituted heteroarylene. In embodiments,L¹⁰⁴ is unsubstituted phenylene.

In embodiments, L¹⁰⁰ is -L¹⁰¹-O—CH(N₃)-L¹⁰³-L¹⁰⁴-L¹⁰⁵-.

In embodiments, L¹⁰¹, L¹⁰³, L¹⁰⁴, and L¹⁰⁵ are independently a bond,—NH—, —O—, —C(O)—, —C(O)NH—, —NHC(O)—, —NHC(O)NH—, —C(O)O—, —OC(O)—,substituted or unsubstituted alkylene (e.g., —CH(OH)— or —C(CH₂)—),substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene.

In embodiments, L¹⁰¹ is independently a substituted or unsubstitutedC₁-C₄ alkylene or substituted or unsubstituted 8 to 20 memberedheteroalkylene; L¹⁰³ is independently a bond or substituted orunsubstituted 2 to 10 membered heteroalkylene; L¹⁰⁴ is independently abond, substituted or unsubstituted 4 to 18 membered heteroalkylene, orsubstituted or unsubstituted phenylene; and L¹⁰⁵ is independently bondor substituted or unsubstituted 4 to 18 membered heteroalkylene. Inembodiments, L¹⁰¹ is independently a substituted or unsubstituted C₁-C₄alkylene or substituted or unsubstituted 8 to 20 memberedheteroalkylene. In embodiments, L¹⁰¹ is independently an oxo-substitutedC₁-C₄ alkylene or an oxo-substituted 8 to 20 membered heteroalkylene. Inembodiments, L¹⁰³ is independently a bond or substituted orunsubstituted 2 to 10 membered heteroalkylene. In embodiments, L¹⁰³ isindependently a bond or an unsubstituted 2 to 10 memberedheteroalkylene. In embodiments, L¹⁰⁴ is independently a bond,substituted or unsubstituted 4 to 18 membered heteroalkylene, orsubstituted or unsubstituted phenylene. In embodiments, L¹⁰⁵ isindependently a bond or substituted or unsubstituted 4 to 18 memberedheteroalkylene. In embodiments, L¹⁰⁵ is independently a bond or anoxo-substituted 4 to 18 membered heteroalkylene. In embodiments, L¹⁰⁵ isindependently a bond or an unsubstituted 4 to 18 memberedheteroalkylene.

In embodiments, L¹⁰⁰ is -L¹⁰¹-O—CH(N₃)—CH₂—O-L¹⁰⁴-L¹⁰⁵-.

In embodiments, L¹⁰¹ and L¹⁰⁵ are independently a bond, —NH—, —O—,—C(O)—, —C(O)NH—, —NHC(O)—, —NHC(O)NH—, —C(O)O—, —OC(O)—, substituted orunsubstituted alkylene (e.g., —CH(OH)— or —C(CH₂)—), substituted orunsubstituted heteroalkylene, substituted or unsubstitutedcycloalkylene, substituted or unsubstituted heterocycloalkylene,substituted or unsubstituted arylene, or substituted or unsubstitutedheteroarylene; and L¹⁰⁴ is unsubstituted phenylene. In embodiments, L¹⁰¹and L¹⁰⁵ are independently a bond, —NH—, —O—, —C(O)—, —C(O)NH—,—NHC(O)—, —NHC(O)NH—, —C(O)O—, —OC(O)—, substituted or unsubstitutedalkylene (e.g., —CH(OH)— or —C(CH₂)—), substituted or unsubstitutedheteroalkylene, substituted or unsubstituted cycloalkylene, substitutedor unsubstituted heterocycloalkylene, substituted or unsubstitutedarylene, or substituted or unsubstituted heteroarylene. In embodiments,L¹⁰¹ is oxo-substituted heteroalkylene. In embodiments, L¹⁰⁴ isunsubstituted phenylene. In embodiments, L¹⁰⁵ is oxo-substitutedheteroalkylene.

In embodiments, L¹⁰⁰ is -L¹⁰¹-SS-L¹⁰³-L¹⁰⁴-L¹⁰⁵-;

In embodiments, L¹⁰¹, L¹⁰⁴, and L¹⁰⁵ are independently a bond, —NH—,—O—, —C(O)—, —C(O)NH—, —NHC(O)—, —NHC(O)NH—, —C(O)O—, —OC(O)—,substituted or unsubstituted alkylene (e.g., —CH(OH)— or —C(CH₂)—),substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene; and L¹⁰³ is a bond orunsubstituted phenylene.

In embodiments, L¹⁰¹ is independently a substituted or unsubstituted 8to 20 membered heteroalkylene; L¹⁰³ is independently a bond orsubstituted or unsubstituted phenylene; L¹⁰⁴ is independently a bond orsubstituted or unsubstituted 4 to 18 membered heteroalkylene; and L¹⁰⁵is independently a bond or substituted or unsubstituted 4 to 18 memberedheteroalkylene. In embodiments, L¹⁰¹ is independently a substituted orunsubstituted 8 to 20 membered heteroalkylene. In embodiments, L¹⁰¹ isindependently an oxo-substituted 8 to 20 membered heteroalkylene. Inembodiments, L¹⁰³ is independently a bond. In embodiments, L¹⁰³ isindependently a substituted phenylene. In embodiments, L¹⁰³ isindependently an unsubstituted phenylene. In embodiments, L¹⁰³ isindependently

In embodiments, L¹⁰⁴ is independently a bond, or substituted orunsubstituted 4 to 18 membered heteroalkylene. In embodiments, L¹⁰⁴ isindependently a bond or an oxo-substituted 4 to 18 memberedheteroalkylene. In embodiments, L¹⁰⁵ is independently a bond orsubstituted or unsubstituted 4 to 18 membered heteroalkylene. Inembodiments, L¹⁰⁵ is independently a bond or an unsubstituted 4 to 18membered heteroalkylene.

In embodiments, L¹⁰⁰ is -L¹⁰¹-SS—C(CH₃)₂-L¹⁰⁴-L¹⁰⁵-.

In embodiments, L¹⁰¹, L¹⁰⁴, and L¹⁰⁵ are independently a bond, —NH—,—O—, —C(O)—, —C(O)NH—, —NHC(O)—, —NHC(O)NH—, —C(O)O—, —OC(O)—,substituted or unsubstituted alkylene (e.g., —CH(OH)— or —C(CH₂)—),substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene.

In embodiments, L¹⁰¹ is independently a substituted or unsubstituted 8to 20 membered heteroalkylene; L¹⁰⁴ is independently a bond orsubstituted or unsubstituted 4 to 18 membered heteroalkylene; and L¹⁰⁵is independently bond or substituted or unsubstituted 4 to 18 memberedheteroalkylene. In embodiments, L¹⁰¹ is independently a substituted orunsubstituted 8 to 20 membered heteroalkylene. In embodiments, L¹⁰¹ isindependently an oxo-substituted 8 to 20 membered heteroalkylene. Inembodiments, L¹⁰⁴ is independently a bond, or substituted orunsubstituted 4 to 18 membered heteroalkylene. In embodiments, L¹⁰⁴ isindependently a bond or an oxo-substituted 4 to 18 memberedheteroalkylene. In embodiments, L¹⁰⁵ is independently a bond orsubstituted or unsubstituted 4 to 18 membered heteroalkylene. Inembodiments, L¹⁰⁵ is independently a bond or an unsubstituted 4 to 18membered heteroalkylene.

In embodiments, L¹⁰⁰ is -(L¹⁰¹)-SS-(L¹⁰³)-(L¹⁰⁴)-(L¹⁰⁵)-. L¹⁰¹, L¹⁰³,L¹⁰⁴, and L¹⁰⁵ are as described herein. In embodiments, L¹⁰⁰ is-(L¹⁰¹)-OCH(R¹⁰²)—SS-(L¹⁰³)-(L¹⁰⁴)-(L¹⁰⁵)-. L¹⁰¹, L¹⁰⁴, and L¹⁰⁵ are asdescribed herein.

In embodiments, L¹⁰⁰ is -L¹⁰¹-CH(OH)—CH(OH)-L¹⁰⁴-L¹⁰⁵-.

In embodiments, L¹⁰¹, L¹⁰⁴, and L¹⁰⁵ are independently a bond, —NH—,—O—, —C(O)—, —C(O)NH—, —NHC(O)—, —NHC(O)NH—, —C(O)O—, —OC(O)—,substituted or unsubstituted alkylene (e.g., —CH(OH)— or —C(CH₂)—),substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene.

In embodiments, L¹⁰¹ is independently a substituted or unsubstituted 3to 10 membered heteroalkylene; L¹⁰⁴ is independently a bond orsubstituted or unsubstituted 2 to 10 membered heteroalkylene; and L¹⁰⁵is independently bond or substituted or unsubstituted 2 to 10 memberedheteroalkylene. In embodiments, L¹⁰¹ is independently a substituted orunsubstituted 3 to 10 membered heteroalkylene. In embodiments, L¹⁰¹ isindependently oxo-substituted 3 to 10 membered heteroalkylene. Inembodiments, L¹⁰⁴ is independently a bond or substituted orunsubstituted 2 to 10 membered heteroalkylene. In embodiments, L¹⁰⁴ isindependently a bond or oxo-substituted 2 to 10 membered heteroalkylene.In embodiments, L¹⁰⁵ is independently a bond or substituted orunsubstituted 2 to 10 membered heteroalkylene. In embodiments, L¹⁰⁵ isindependently a bond or unsubstituted 2 to 10 membered heteroalkylene.

In embodiments, L¹⁰⁰ is -L¹⁰¹-C(CH₂)-L¹⁰³-L¹⁰⁴-L¹⁰⁵-.

In embodiments, L¹⁰¹, L¹⁰³, L¹⁰⁴, and L¹⁰⁵ are independently a bond,—NH—, —O—, —C(O)—, —C(O)NH—, —NHC(O)—, —NHC(O)NH—, —C(O)O—, —OC(O)—,substituted or unsubstituted alkylene (e.g., —CH(OH)— or —C(CH₂)—),substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene.

In embodiments, L¹⁰¹ is independently a substituted or unsubstituted 3to 10 membered heteroalkylene; L¹⁰³ is independently a bond orsubstituted or unsubstituted 2 to 10 membered heteroalkylene; L¹⁰⁴ isindependently a bond or substituted or unsubstituted 2 to 10 memberedheteroalkylene; and L¹⁰⁵ is independently bond or substituted orunsubstituted 2 to 10 membered heteroalkylene. In embodiments, L¹⁰¹ isindependently a substituted or unsubstituted 3 to 10 memberedheteroalkylene. In embodiments, L¹⁰¹ is independently oxo-substituted 3to 10 membered heteroalkylene. In embodiments, L¹⁰³ is independently abond or substituted or unsubstituted 2 to 10 membered heteroalkylene. Inembodiments, L¹⁰³ is independently a bond or unsubstituted 2 to 10membered heteroalkylene. In embodiments, L¹⁰⁴ is independently a bond orsubstituted or unsubstituted 2 to 10 membered heteroalkylene. Inembodiments, L¹⁰⁴ is independently a bond or unsubstituted 2 to 10membered heteroalkylene. In embodiments, L¹⁰⁵ is independently a bond orsubstituted or unsubstituted 2 to 10 membered heteroalkylene. Inembodiments, L¹⁰⁵ is independently a bond or unsubstituted 2 to 10membered heteroalkylene.

In embodiments, L¹⁰⁰ is -L¹⁰¹-L¹⁰²-L¹⁰³-L¹⁰⁴-L¹⁰⁵-.

In embodiments, L¹⁰¹, L¹⁰³, and L¹⁰⁵ are independently a bond, —NH—,—O—, —C(O)—, —C(O)NH—, —NHC(O)—, —NHC(O)NH—, —C(O)O—, —OC(O)—, —N═N—,substituted or unsubstituted alkylene (e.g., —CH(OH)— or —C(CH₂)—),substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene; and L¹⁰² and L¹⁰⁴ aresubstituted or unsubstituted phenylene. In embodiments, L¹⁰¹, L¹⁰³, andL¹⁰⁵ are independently a bond, —NH—, —O—, —C(O)—, —C(O)NH—, —NHC(O)—,—NHC(O)NH—, —C(O)O—, —OC(O)—, —N═N—, substituted or unsubstitutedalkylene (e.g., —CH(OH)— or —C(CH₂)—), substituted or unsubstitutedheteroalkylene, substituted or unsubstituted cycloalkylene, substitutedor unsubstituted heterocycloalkylene, substituted or unsubstitutedarylene, or substituted or unsubstituted heteroarylene. In embodiments,L¹⁰³ is independently —N═N—. In embodiments, L¹⁰² is independently asubstituted phenylene. In embodiments, L¹⁰² is independently anunsubstituted phenylene. In embodiments, L¹⁰² is independently

In embodiments, L¹⁰⁴ is independently a substituted phenylene. Inembodiments, L¹⁰⁴ is independently an unsubstituted phenylene. Inembodiments, L¹⁰⁴ is independently

In embodiments, L¹⁰⁴ is independently

In embodiments, L¹⁰¹ is independently a substituted or unsubstituted 3to 10 membered heteroalkylene; L¹⁰³ is independently a bond orsubstituted or unsubstituted 2 to 10 membered heteroalkylene; L¹⁰⁵ isindependently bond or substituted or unsubstituted 5 to 16 memberedheteroalkylene; and L¹⁰² and L¹⁰⁴ are substituted or unsubstitutedphenylene. In embodiments, L¹⁰¹ is independently a substituted orunsubstituted 3 to 10 membered heteroalkylene. In embodiments, L¹⁰¹ isindependently oxo-substituted 3 to 10 membered heteroalkylene. Inembodiments, L¹⁰³ is independently a bond or substituted orunsubstituted 2 to 10 membered heteroalkylene. In embodiments, L¹⁰³ isindependently a bond or unsubstituted 2 to 10 membered heteroalkylene.In embodiments, L¹⁰³ is independently —N═N—. In embodiments, L¹⁰⁵ isindependently bond or substituted or unsubstituted 5 to 16 memberedheteroalkylene. In embodiments, L¹⁰⁵ is independently bond orunsubstituted 5 to 16 membered heteroalkylene. In embodiments, L¹⁰² isindependently a substituted phenylene. In embodiments, L¹⁰² isindependently an unsubstituted phenylene. In embodiments, L¹⁰⁴ isindependently a substituted phenylene. In embodiments, L¹⁰⁴ isindependently an unsubstituted phenylene.

In embodiments, L¹⁰⁰ is -L¹⁰¹-O—CH(R¹⁰²)-L¹⁰³-L¹⁰⁴-L¹⁰⁵-.

In embodiments, L¹⁰¹, L¹⁰³, L¹⁰⁴, and L¹⁰⁵ are independently a bond,—NH—, —O—, —C(O)—, —C(O)NH—, —NHC(O)—, —NHC(O)NH—, —C(O)O—, —OC(O)—,substituted or unsubstituted alkylene (e.g., —CH(OH)— or —C(CH₂)—),substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene; and R¹⁰² isR^(102B)-substituted C₁-C₄ alkyl. In embodiments, L¹⁰¹, L¹⁰⁴, and L¹⁰⁵are independently a bond, —NH—, —O—, —C(O)—, —C(O)NH—, —NHC(O)—,—NHC(O)NH—, —C(O)O—, —OC(O)—, substituted or unsubstituted alkylene(e.g., —CH(OH)— or —C(CH₂)—), substituted or unsubstitutedheteroalkylene, substituted or unsubstituted cycloalkylene, substitutedor unsubstituted heterocycloalkylene, substituted or unsubstitutedarylene, or substituted or unsubstituted heteroarylene. In embodiments,R¹⁰² is R^(102B)-substituted C₁-C₄ alkyl. R^(102B) is independently oxo,halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂,—SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂,—NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃, —OCF₃, —OCBr₃, —OCI₃,—OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —N₃, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl. In embodiments, R^(102B) is independently —CN.

In embodiments, L¹⁰¹, L¹⁰³, L¹⁰⁴, and L¹⁰⁵ are independently a bond,—NH—, —O—, —C(O)—, —C(O)NH—, —NHC(O)—, —NHC(O)NH—, —C(O)O—, —OC(O)—,substituted or unsubstituted alkylene (e.g., —CH(OH)— or —C(CH₂)—),substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene; and R¹⁰² isR^(102B)-substituted C₁-C₄ alkyl. In embodiments, L¹⁰¹, L¹⁰⁴, and L¹⁰⁵are independently a bond, —NH—, —O—, —C(O)—, —C(O)NH—, —NHC(O)—,—NHC(O)NH—, —C(O)O—, —OC(O)—, substituted or unsubstituted alkylene(e.g., —CH(OH)— or —C(CH₂)—), substituted or unsubstitutedheteroalkylene, substituted or unsubstituted cycloalkylene, substitutedor unsubstituted heterocycloalkylene, substituted or unsubstitutedarylene, or substituted or unsubstituted heteroarylene. In embodiments,R¹⁰² is R^(102B)-substituted C₁-C₄ alkyl. In embodiments, R^(102B) isindependently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CN, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —N₃,unsubstituted alkyl, unsubstituted heteroalkyl, unsubstitutedcycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, orunsubstituted heteroaryl. In embodiments, R^(102B) is independently —CN.

In embodiments, L¹⁰¹, L¹⁰³, L¹⁰⁴, and L¹⁰⁵ are independently a bond,—NH—, —O—, —C(O)—, —C(O)NH—, —NHC(O)—, —NHC(O)NH—, —C(O)O—, —OC(O)—,substituted or unsubstituted alkylene (e.g., —CH(OH)— or —C(CH₂)—),substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene; and R¹⁰² isR^(102B)-substituted C₁-C₄ alkyl. In embodiments, L¹⁰¹, L¹⁰⁴, and L¹⁰⁵are independently a bond, —NH—, —O—, —C(O)—, —C(O)NH—, —NHC(O)—,—NHC(O)NH—, —C(O)O—, —OC(O)—, substituted or unsubstituted alkylene(e.g., —CH(OH)— or —C(CH₂)—), substituted or unsubstitutedheteroalkylene, substituted or unsubstituted cycloalkylene, substitutedor unsubstituted heterocycloalkylene, substituted or unsubstitutedarylene, or substituted or unsubstituted heteroarylene. In embodiments,R¹⁰² is R^(102B)-substituted C₁-C₄ alkyl. In embodiments, R^(102B) isindependently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CN, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —N₃,unsubstituted alkyl (e.g., C₁-C₈, C₁-C₆, or C₁-C₄), unsubstitutedheteroalkyl (e.g., 2 to 10 membered, 2 to 8 membered, 2 to 6 membered,or 2 to 4 membered), unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, orC₅-C₆), unsubstituted heterocycloalkyl (e.g., 3 to 8, 3 to 6, or 5 to 6membered), unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, or phenyl), orunsubstituted heteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered). Inembodiments, R^(102B) is independently —CN.

In embodiments, L¹⁰⁰ is -L¹⁰¹-O—CH(CH₂R¹⁰²)-L¹⁰³-L¹⁰⁴-L¹⁰⁵-.

In embodiments, L¹⁰¹, L¹⁰³, L¹⁰⁴, and L¹⁰⁵ are independently a bond,—NH—, —O—, —C(O)—, —C(O)NH—, —NHC(O)—, —NHC(O)NH—, —C(O)O—, —OC(O)—,substituted or unsubstituted alkylene (e.g., —CH(OH)— or —C(CH₂)—),substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene; and R¹⁰² is independentlyoxo, hydrogen, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CN, —OH, —NH₂, —COOH,—CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂,—NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃, —OCF₃, —OCBr₃,—OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —N₃, unsubstituted alkyl (e.g.,C₁-C₈, C₁-C₆, or C₁-C₄), unsubstituted heteroalkyl (e.g., 2 to 10membered, 2 to 8 membered, 2 to 6 membered, or 2 to 4 membered),unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, or C₅-C₆), unsubstitutedheterocycloalkyl (e.g., 3 to 8, 3 to 6, or 5 to 6 membered),unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, or phenyl), or unsubstitutedheteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered). In embodiments,L¹⁰¹, L¹⁰⁴, and L¹⁰⁵ are independently a bond, —NH—, —O—, —C(O)—,—C(O)NH—, —NHC(O)—, —NHC(O)NH—, —C(O)O—, —OC(O)—, substituted orunsubstituted alkylene (e.g., —CH(OH)— or —C(CH₂)—), substituted orunsubstituted heteroalkylene, substituted or unsubstitutedcycloalkylene, substituted or unsubstituted heterocycloalkylene,substituted or unsubstituted arylene, or substituted or unsubstitutedheteroarylene. In embodiments, R¹⁰² is independently oxo, hydrogen,halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂,—SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂,—NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃, —OCF₃, —OCBr₃, —OCI₃,—OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —N₃, unsubstituted alkyl (e.g., C₁-C₈,C₁-C₆, or C₁-C₄), unsubstituted heteroalkyl (e.g., 2 to 10, 2 to 8, 2 to6, or 2 to 4 membered), unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, orC₅-C₆), unsubstituted heterocycloalkyl (e.g., 3 to 8, 3 to 6, or 5 to 6membered), unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, or phenyl), orunsubstituted heteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered). Inembodiments, R¹⁰² is independently —CN.

In embodiments, L¹⁰¹ is independently a substituted or unsubstituted 3to 10 membered heteroalkylene; L¹⁰³ is independently a bond orsubstituted or unsubstituted 5 to 16 membered heteroalkylene; L¹⁰⁴ isindependently a bond or substituted or unsubstituted 5 to 16 memberedheteroalkylene; L¹⁰⁵ is independently bond or substituted orunsubstituted 5 to 16 membered heteroalkylene; and R¹⁰² is independently—CN. In embodiments, L¹⁰¹ is independently a substituted orunsubstituted 3 to 10 membered heteroalkylene. In embodiments, L¹⁰¹ isindependently oxo-substituted 3 to 10 membered heteroalkylene. Inembodiments, L¹⁰³ is independently a bond or substituted orunsubstituted 5 to 16 membered heteroalkylene. In embodiments, L¹⁰³ isindependently a bond or unsubstituted 5 to 16 membered heteroalkylene.In embodiments, L¹⁰⁴ is independently a bond or substituted orunsubstituted 5 to 16 membered heteroalkylene. In embodiments, L¹⁰⁴ isindependently a bond or unsubstituted 5 to 16 membered heteroalkylene.In embodiments, L¹⁰⁵ is independently a bond or substituted orunsubstituted 5 to 16 membered heteroalkylene. In embodiments, L¹⁰⁵ isindependently a bond or unsubstituted 5 to 16 membered heteroalkylene.In embodiments, R¹⁰² is independently —CN.

In embodiments, L¹⁰⁰ is -L¹⁰¹-O—CH(CH₂R¹⁰²)—CH₂—O-L¹⁰⁴-L¹⁰⁵-.

In embodiments, L¹⁰¹, L¹⁰⁴, and L¹⁰⁵ are independently a bond, —NH—,—O—, —C(O)—, —C(O)NH—, —NHC(O)—, —NHC(O)NH—, —C(O)O—, —OC(O)—,substituted or unsubstituted alkylene (e.g., —CH(OH)— or —C(CH₂)—),substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene; and R¹⁰² is independentlyoxo, hydrogen, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CN, —OH, —NH₂, —COOH,—CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂,—NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃, —OCF₃, —OCBr₃,—OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —N₃, unsubstituted alkyl (e.g.,C₁-C₈, C₁-C₆, or C₁-C₄), unsubstituted heteroalkyl (e.g., 2 to 10, 2 to8, 2 to 6, or 2 to 4 membered), unsubstituted cycloalkyl (e.g., C₃-C₈,C₃-C₆, or C₅-C₆), unsubstituted heterocycloalkyl (e.g., 3 to 8, 3 to 6,or 5 to 6 membered), unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, or phenyl),or unsubstituted heteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered).In embodiments, L¹⁰¹, L¹⁰⁴, and L¹⁰⁵ are independently a bond, —NH—,—O—, —C(O)—, —C(O)NH—, —NHC(O)—, —NHC(O)NH—, —C(O)O—, —OC(O)—,substituted or unsubstituted alkylene (e.g., —CH(OH)— or —C(CH₂)—),substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene. In embodiments, R¹⁰² isindependently oxo, hydrogen, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CN,—OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCCl₃, —OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —N₃,unsubstituted alkyl (e.g., C₁-C₈, C₁-C₆, or C₁-C₄), unsubstitutedheteroalkyl (e.g., 2 to 10, 2 to 8, 2 to 6, or 2 to 4 membered),unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, or C₅-C₆), unsubstitutedheterocycloalkyl (e.g., 3 to 8, 3 to 6, or 5 to 6 membered),unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, or phenyl), or unsubstitutedheteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered). In embodiments,R¹⁰² is independently —CN.

In embodiments, L¹⁰⁰ is -L¹⁰¹-O—CH(CH₂CN)—CH₂—O-L¹⁰⁴-L¹⁰⁵-.

In embodiments, L¹⁰¹, L¹⁰⁴, and L¹⁰⁵ are independently a bond, —NH—,—O—, —C(O)—, —C(O)NH—, —NHC(O)—, —NHC(O)NH—, —C(O)O—, —OC(O)—,substituted or unsubstituted alkylene (e.g., —CH(OH)— or —C(CH₂)—),substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene.

In embodiments, L¹⁰¹ is independently a bond, —NH—, —O—, —C(O)—,—C(O)NH—, —NHC(O)—, —NHC(O)NH—, —C(O)O—, —OC(O)—, substituted orunsubstituted alkylene, substituted or unsubstituted heteroalkylene,substituted or unsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene.

In embodiments, L¹⁰¹ is a bond, —NH—, —S—, —O—, —C(O)—, —C(O)O—,—OC(O)—, —NHC(O)—, —C(O)NH—, —NHC(O)NH—, —NHC(NH)NH—, —C(S)—,substituted or unsubstituted alkylene (e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₈,C₁-C₆, or C₁-C₄), substituted or unsubstituted heteroalkylene (e.g., 2to 20 membered, 8 to 20 membered, 2 to 10 membered, 3 to 10 membered, 2to 8 membered, 2 to 6 membered, or 2 to 4 membered), substituted orunsubstituted cycloalkylene (e.g., C₃-C₈, C₃-C₆, or C₅-C₆), substitutedor unsubstituted heterocycloalkylene (e.g., 3 to 8, 3 to 6, or 5 to 6membered), substituted or unsubstituted arylene (e.g., C₆-C₁₀, C₁₀, orphenylene), or substituted or unsubstituted heteroarylene (e.g., 5 to10, 5 to 9, or 5 to 6 membered).

In embodiments, L¹⁰¹ is a bond, —NH—, —NR¹⁰¹—, —S—, —O—, —C(O)—,—C(O)O—, —OC(O)—, —NHC(O)—, —C(O)NH—, —NHC(O)NH—, —NHC(NH)NH—, —C(S)—,R¹⁰¹-substituted or unsubstituted alkylene (e.g., C₁-C₂₀, C₁₀-C₂₀,C₁-C₈, C₁-C₆, or C₁-C₄), R¹⁰¹-substituted or unsubstitutedheteroalkylene (e.g., 2 to 20 membered, 8 to 20 membered, 2 to 10membered, 3 to 10 membered, 2 to 8 membered, 2 to 6 membered, or 2 to 4membered), R¹⁰¹-substituted or unsubstituted cycloalkylene (e.g., C₃-C₈,C₃-C₆, or C₅-C₆), R¹⁰¹-substituted or unsubstituted heterocycloalkylene(e.g., 3 to 8, 3 to 6, or 5 to 6 membered), R¹⁰¹-substituted orunsubstituted arylene (e.g., C₆-C₁₀, C₁₀, or phenylene), orR¹⁰¹-substituted or unsubstituted heteroarylene (e.g., 5 to 10, 5 to 9,or 5 to 6 membered). In embodiments, L¹⁰¹ is a bond. In embodiments,L¹⁰¹ is —NH—. In embodiments, L¹⁰¹ is —NR¹⁰¹—. In embodiments, L¹⁰¹ is-5-. In embodiments, L¹⁰¹ is —O—. In embodiments, L¹⁰¹ is —C(O)—. Inembodiments, L¹⁰¹ is —C(O)O—. In embodiments, L¹⁰¹ is —OC(O)—. Inembodiments, L¹⁰¹ is —NHC(O)—. In embodiments, L¹⁰¹ is —C(O)NH—. Inembodiments, L¹⁰¹ is —NHC(O)NH—. In embodiments, L¹⁰¹ is —NHC(NH)NH—. Inembodiments, Li¹⁰¹ is —C(S)—. In embodiments, L¹⁰¹ is R¹⁰¹-substitutedor unsubstituted C₁-C₂₀ alkylene. In embodiments, L¹⁰¹ isR¹⁰¹-substituted or unsubstituted 2 to 20 membered heteroalkylene. Inembodiments, L¹⁰¹ is R¹⁰¹-substituted or unsubstituted 3 to 10 memberedheteroalkylene. In embodiments, L¹⁰¹ is R¹⁰¹-substituted orunsubstituted C₃-C₈ cycloalkylene. In embodiments, Li¹⁰¹ isR¹⁰¹-substituted or unsubstituted 3 to 8 membered heterocycloalkylene.In embodiments, Li¹⁰¹ is R¹⁰¹-substituted or unsubstituted C₆-C₁₀arylene. In embodiments, L¹⁰¹ is R¹⁰¹-substituted or unsubstituted 5 to10 membered heteroarylene.

In embodiments, L¹⁰¹ is a bond, —NH—, —NR¹⁰¹—, —S—, —O—, —C(O)—,—C(O)O—, —OC(O)—, —NHC(O)—, —C(O)NH—, —NHC(O)NH—, —NHC(NH)NH—, —C(S)—,—CH(OH)—, or —C(CH₂)—. In embodiments, L¹⁰¹ is a bond. In embodiments,L¹⁰¹ is —NH—. In embodiments, L¹⁰¹ is —NR¹⁰¹—. In embodiments, L¹⁰¹ is—S—. In embodiments, L¹⁰¹ is —O—. In embodiments, L¹⁰¹ is —C(O)—. Inembodiments, L¹⁰¹ is —C(O)O—. In embodiments, L¹⁰¹ is —OC(O)—. Inembodiments, L¹⁰¹ is —NHC(O)—. In embodiments, L¹⁰¹ is —C(O)NH—. Inembodiments, L¹⁰¹ is —NHC(O)NH—. In embodiments, L¹⁰¹ is —NHC(NH)NH—. Inembodiments, L¹⁰¹ is —C(S)—. In embodiments, L¹⁰¹ is —CH(OH)—. Inembodiments, L¹⁰¹ is —C(CH₂)—.

In embodiments, L¹⁰¹ is —(CH₂CH₂O)_(b)—. In embodiments, L¹⁰¹ is—CCCH₂(OCH₂CH₂)_(a)—NHC(O)—(CH₂), (OCH₂CH₂)_(b)—. In embodiments, L¹⁰¹is —CHCHCH₂—NHC(O)—(CH₂), (OCH₂CH₂)_(b)—. In embodiments, L¹⁰¹ is—CCCH₂—NHC(O)—(CH₂), (OCH₂CH₂)_(b)—. In embodiments, L¹⁰¹ is —CCCH₂—.The symbol a is an integer from 0 to 8. In embodiments, a is 1. Inembodiments, a is 0. The symbol b is an integer from 0 to 8. Inembodiments, b is 0. In embodiments, b is 1 or 2. In embodiments, b isan integer from 2 to 8. In embodiments, b is 1. The symbol c is aninteger from 0 to 8. In embodiments, c is 0. In embodiments, c is 1. Inembodiments, c is 2. In embodiments, c is 3.

R¹⁰¹ is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CN, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —N₃,R^(101A)-substituted or unsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀,C₁-C₈, C₁-C₆, or C₁-C₄), R^(101A)-substituted or unsubstitutedheteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to 6, or 2 to 4membered), R^(101A)-substituted or unsubstituted cycloalkyl (e.g.,C₃-C₈, C₃-C₆, or C₅-C₆), R^(101A)-substituted or unsubstitutedheterocycloalkyl (e.g., 3 to 8, 3 to 6, or 5 to 6 membered),R^(101A)-substituted or unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, orphenyl), or R^(101A)-substituted or unsubstituted heteroaryl (e.g., 5 to10, 5 to 9, or 5 to 6 membered).

In embodiments, R¹⁰¹ is independently —NH₂. In embodiments, R¹⁰¹ isindependently —OH. In embodiments, R¹⁰¹ is independently halogen. Inembodiments, R¹⁰¹ is independently —CN. In embodiments, R¹⁰¹ isindependently oxo. In embodiments, R¹⁰¹ is independently —CF₃. Inembodiments, R¹⁰¹ is independently —COOH. In embodiments, R¹⁰¹ isindependently —CONH₂. In embodiments, R¹⁰¹ is independently —F. Inembodiments, R¹⁰¹ is independently —Cl. In embodiments, R¹⁰¹ isindependently —Br. In embodiments, R¹⁰¹ is independently —I.

R^(101A) is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CN,—OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCCl₃, —OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —N₃,R^(101B)-substituted or unsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀,C₁-C₈, C₁-C₆, or C₁-C₄), R^(101B)-substituted or unsubstitutedheteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to 6, or 2 to 4membered), R^(101B)-substituted or unsubstituted cycloalkyl (e.g.,C₃-C₈, C₃-C₆, or C₅-C₆), R^(101B)-substituted or unsubstitutedheterocycloalkyl (e.g., 3 to 8, 3 to 6, or 5 to 6 membered),R^(101B)-substituted or unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, orphenyl), or R^(101B)-substituted or unsubstituted heteroaryl (e.g., 5 to10, 5 to 9, or 5 to 6 membered).

R^(101B) is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CN,—OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCCl₃, —OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —N₃,unsubstituted alkyl (e.g., C₁—C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄),unsubstituted heteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to6, or 2 to 4 membered), unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, orC₅-C₆), unsubstituted heterocycloalkyl (e.g., 3 to 8, 3 to 6, or 5 to 6membered), unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, or phenyl), orunsubstituted heteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered).

In embodiments, L¹⁰² is independently a bond, —NH—, —O—, —C(O)—,—C(O)NH—, —NHC(O)—, —NHC(O)NH—, —C(O)O—, —OC(O)—, substituted orunsubstituted alkylene, substituted or unsubstituted heteroalkylene,substituted or unsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene.

In embodiments, L¹⁰² is a bond, —NH—, —S—, —O—, —C(O)—, —C(O)O—,—OC(O)—, —NHC(O)—, —C(O)NH—, —NHC(O)NH—, —NHC(NH)NH—, —C(S)—, —SS—,substituted or unsubstituted alkylene (e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₈,C₁-C₆, or C₁-C₄), substituted or unsubstituted heteroalkylene (e.g., 2to 20, 8 to 20, 2 to 10, 2 to 8, 2 to 6, or 2 to 4 membered),substituted or unsubstituted cycloalkylene (e.g., C₃-C₈, C₃-C₆, orC₅-C₆), substituted or unsubstituted heterocycloalkylene (e.g., 3 to 8,3 to 6, or 5 to 6 membered), substituted or unsubstituted arylene (e.g.,C₆-C₁₀, C₁₀, or phenylene), or substituted or unsubstitutedheteroarylene (e.g., 5 to 10, 5 to 9, or 5 to 6 membered). Inembodiments, L¹⁰² is a bond, —NH—, —S—, —O—, —C(O)—, —C(O)O—, —OC(O)—,—NHC(O)—, —C(O)NH—, —NHC(O)NH—, —NHC(NH)NH—, —C(S)—, substituted orunsubstituted alkylene (e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄),substituted or unsubstituted heteroalkylene (e.g., 2 to 20, 8 to 20, 2to 10, 2 to 8, 2 to 6, or 2 to 4 membered), substituted or unsubstitutedcycloalkylene (e.g., C₃-C₈, C₃-C₆, or C₅-C₆), substituted orunsubstituted heterocycloalkylene (e.g., 3 to 8, 3 to 6, or 5 to 6membered), substituted or unsubstituted arylene (e.g., C₆-C₁₀, C₁₀, orphenylene), or substituted or unsubstituted heteroarylene (e.g., 5 to10, 5 to 9, or 5 to 6 membered).

In embodiments, L¹⁰² is a bond, —NH—, —OC(—SR′° °)(R^(102a))—,—OC(—SSR¹⁰²)(R^(102a))—, —OC(—SCN)(R^(102a))—, —OC(N₃)(R^(102a))—,—OCH(R¹⁰²)—, —OCH(CH₂R¹⁰²)—, —OCH(CH₂CN)—, —S—, —O—, —C(O)—, —C(O)O—,—OC(O)—, —NHC(O)—, —C(O)NH—, —NHC(O)NH—, —NHC(NH)NH—, —C(S)—, —SS—,R¹⁰²-substituted or unsubstituted alkylene (e.g., C₁-C₂₀, C₁₀-C₂₀,C₁-C₈, C₁-C₆, or C₁-C₄), R¹⁰²-substituted or unsubstitutedheteroalkylene (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to 6, or 2 to4 membered), R¹⁰²-substituted or unsubstituted cycloalkylene (e.g.,C₃-C₈, C₃-C₆, or C₅-C₆), R¹⁰²-substituted or unsubstitutedheterocycloalkylene (e.g., 3 to 8, 3 to 6, or 5 to 6 membered),R¹⁰²-substituted or unsubstituted arylene (e.g., C₆-C₁₀, C₁₀, orphenylene), or R¹⁰²-substituted or unsubstituted heteroarylene (e.g., 5to 10, 5 to 9, or 5 to 6 membered). In embodiments, L¹⁰² is a bond. Inembodiments, L¹⁰² is —NH—. In embodiments, L¹⁰² is—OC(—SR¹⁰⁰)(R^(102a))—. In embodiments, L¹⁰² is —OC(—SSR¹⁰²)(R^(102a))—.In embodiments, L¹⁰² is —OC(—SCN)(R^(102a))—. In embodiments, L¹⁰² is—OC(N₃)(R^(102a))—. In embodiments, L¹⁰² is —OC(—SR¹⁰⁰)(CH₃)—. Inembodiments, L¹⁰² is —OC(—SSR¹⁰²)(CH₃)—. In embodiments, L¹⁰² is—OC(—SCN)(CH₃)—. In embodiments, L¹⁰² is —OC(N₃)(CH₃)—. In embodiments,L¹⁰² is —OCH(—SR¹⁰⁰)—. In embodiments, L¹⁰² is —OCH(—SSR¹⁰²)—. Inembodiments, L¹⁰² is —OCH(—SCN)—. In embodiments, L¹⁰² is —OCH(N₃)—. Inembodiments, L¹⁰² is —OCH(R¹⁰²)—. In embodiments, L¹⁰² is—OCH(CH₂R¹⁰²)—. In embodiments, L¹⁰² is —OCH(CH₂CN)—. In embodiments,L¹⁰² is —S—. In embodiments, L¹⁰² is —O—. In embodiments, L¹⁰² is—C(O)—. In embodiments, L¹⁰² is —C(O)O—. In embodiments, L¹⁰² is—OC(O)—. In embodiments, L¹⁰² is —NHC(O)—. In embodiments, L¹⁰² is—C(O)NH—. In embodiments, L¹⁰² is —NHC(O)NH—. In embodiments, L¹⁰² is—NHC(NH)NH—. In embodiments, L¹⁰² is —C(S)—. In embodiments, L¹⁰² is—SS—. In embodiments, L¹⁰² is R¹⁰²-substituted or unsubstituted C₁-C₂₀alkylene. In embodiments, L¹⁰² is R¹⁰²-substituted or unsubstituted 2 to20 membered heteroalkylene. In embodiments, L¹⁰² is R¹⁰²-substituted orunsubstituted C₃-C₈ cycloalkylene. In embodiments, L¹⁰² isR¹⁰²-substituted or unsubstituted 3 to 8 membered heterocycloalkylene.In embodiments, L¹⁰² is R¹⁰²-substituted or unsubstituted C₆-C₁₀arylene. In embodiments, L¹⁰² is R¹⁰²-substituted or unsubstitutedphenylene. In embodiments, L¹⁰² is R¹⁰²-substituted or unsubstituted 5to 10 membered heteroarylene.

In embodiments, L¹⁰² is a bond, —NH—, —OC(—SR¹⁰⁰)(R^(102a))—,—OC(—SSR¹⁰²)(R^(102a))—, —OC(—SCN)(R^(102a))—, —OC(N₃)(R^(102a))—,—OCH(R¹⁰²)—, —OCH(CH₂R¹⁰²)—, —OCH(CH₂CN)—, —S—, —O—, —C(O)—, —C(O)O—,—OC(O)—, —NHC(O)—, —C(O)NH—, —NHC(O)NH—, —NHC(NH)NH—, —C(S)—, —SS—,—CH(OH)—, or —C(CH₂)—. In embodiments, L¹⁰² is a bond. In embodiments,L¹⁰² is —NH—. In embodiments, L¹⁰² is —OC(—SR¹⁰⁰)(R^(102a))—. Inembodiments, L¹⁰² is —OC(—SSR¹⁰²)(R^(102a))—. In embodiments, L¹⁰² is—OC(—SCN)(R^(102a))—. In embodiments, L¹⁰² is —OC(N₃)(R^(102a))—. Inembodiments, L¹⁰² is —OC(—SR¹⁰⁰)(CH₃)—. In embodiments, L¹⁰² is—OC(—SSR¹⁰²)(CH₃)—. In embodiments, L¹⁰² is —OC(—SCN)(CH₃)—. Inembodiments, L¹⁰² is —OC(N₃)(CH₃)—. In embodiments, L¹⁰² is—OCH(—SR¹⁰⁰)—. In embodiments, L¹⁰² is —OCH(—SSR¹⁰²)—. In embodiments,L¹⁰² is —OCH(—SCN)—. In embodiments, L¹⁰² is —OCH(N₃)—. In embodiments,L¹⁰² is —OCH(R¹⁰²)—. In embodiments, L¹⁰² is —OCH(CH₂R¹⁰²)—. Inembodiments, L¹⁰² is —OCH(CH₂CN)—. In embodiments, L¹⁰² is —S—. Inembodiments, L¹⁰² is —O—. In embodiments, L¹⁰² is —C(O)—. Inembodiments, L¹⁰² is —C(O)O—. In embodiments, L¹⁰² is —OC(O)—. Inembodiments, L¹⁰² is —NHC(O)—. In embodiments, L¹⁰² is —C(O)NH—. Inembodiments, L¹⁰² is —NHC(O)NH—. In embodiments, L¹⁰² is —NHC(NH)NH—. Inembodiments, L¹⁰² is —C(S)—. In embodiments, L¹⁰² is —SS—. Inembodiments, L¹⁰² is —CH(OH)—. In embodiments, L¹⁰² is —C(CH₂)—.

R¹⁰⁰ is —SO₃H, —SR¹⁰², or —CN. In embodiments, R¹⁰⁰ is —SR¹⁰². Inembodiments, R¹⁰⁰ is —CN. In embodiments, R¹⁰⁰ is

or In embodiments, R¹⁰⁰ is

In embodiments, R¹⁰⁰ is —SR¹⁰² or —CN. In embodiments, R¹⁰⁰ is —SR¹⁰².In embodiments, R¹⁰⁰ is —CN. In embodiments, R¹⁰⁰ is

R¹⁰² and R^(102a) are independently hydrogen, halogen, —CCl₃, —CBr₃,—CF₃, —CI₃, —CHCl₂, —CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I,—CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCCl₃, —OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, substituted or unsubstituted alkyl(e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄), substituted orunsubstituted heteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to6, or 2 to 4 membered), substituted or unsubstituted cycloalkyl (e.g.,C₃-C₈, C₃-C₆, or C₅-C₆), substituted or unsubstituted heterocycloalkyl(e.g., 3 to 8, 3 to 6, or 5 to 6 membered), substituted or unsubstitutedaryl (e.g., C₆-C₁₀, C₁₀, or phenyl), or substituted or unsubstitutedheteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered).

In embodiments, R¹⁰² is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃,—CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂,—NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH,—NHOH, —OCCl₃, —OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂,—N₃, R^(102B)-substituted or unsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀,C₁-C₈, C₁-C₆, or C₁-C₄), R^(102B)-substituted or unsubstitutedheteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to 6, or 2 to 4membered), R^(102B)-substituted or unsubstituted cycloalkyl (e.g.,C₃-C₈, C₃-C₆, or C₅-C₆), R^(102B)-substituted or unsubstitutedheterocycloalkyl (e.g., 3 to 8, 3 to 6, or 5 to 6 membered),R^(102B)-substituted or unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, orphenyl), or R^(102B)-substituted or unsubstituted heteroaryl (e.g., 5 to10, 5 to 9, or 5 to 6 membered).

In embodiments, R¹⁰² is independently -MB. In embodiments, R¹⁰² isindependently —OH. In embodiments, R¹⁰² is independently halogen. Inembodiments, R¹⁰² is independently —CN. In embodiments, R¹⁰² isindependently oxo. In embodiments, R¹⁰² is independently —CF₃. Inembodiments, R¹⁰² is independently —COOH. In embodiments, R¹⁰² isindependently -COMB. In embodiments, R¹⁰² is independently —F. Inembodiments, R¹⁰² is independently —Cl. In embodiments, R¹⁰² isindependently —Br. In embodiments, R¹⁰² is independently —I.

In embodiments, R¹⁰² is independently unsubstituted alkyl (e.g., C₁-C₂₀,C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄). In embodiments, R¹⁰² is independentlyunsubstituted C₁-C₆ alkyl. In embodiments, R¹⁰² is independentlyunsubstituted C₁-C₄ alkyl. In embodiments, R¹⁰² is independentlyunsubstituted methyl. In embodiments, R¹⁰² is independentlyunsubstituted tert-butyl. In embodiments, R¹⁰² is independentlyhydrogen.

In embodiments, R^(102B) is independently oxo, halogen, —CCl₃, —CBr₃,—CF₃, —CI₃, —CN, —OH, -MB, —COOH, -CONH₂, —NO₂, —SH, —SO₃H, —SO₄H,—SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H,—NHC(O)OH, —NHOH, —OCCl₃, —OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂,—OCHI₂, —OCHF₂, —N₃, substituted or unsubstituted alkyl (e.g., C₁-C₂₀,C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄), substituted or unsubstitutedheteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to 6, or 2 to 4membered), substituted or unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆,or C₅-C₆), substituted or unsubstituted heterocycloalkyl (e.g., 3 to 8,3 to 6, or 5 to 6 membered), substituted or unsubstituted aryl (e.g.,C₆-C₁₀, C₁₀, or phenyl), or substituted or unsubstituted heteroaryl(e.g., 5 to 10, 5 to 9, or 5 to 6 membered). In embodiments, R^(102B) isindependently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CN, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —N₃,R^(102C)-substituted or unsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀,C₁-C₈, C₁-C₆, or C₁-C₄), R^(102C)-substituted or unsubstitutedheteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to 6, or 2 to 4membered), R^(102C)-substituted or unsubstituted cycloalkyl (e.g.,C₃-C₈, C₃-C₆, or C₅-C₆), R^(102C)-substituted or unsubstitutedheterocycloalkyl (e.g., 3 to 8, 3 to 6, or 5 to 6 membered),R^(102C)-substituted or unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, orphenyl), or R^(102C)-substituted or unsubstituted heteroaryl (e.g., 5 to10, 5 to 9, or 5 to 6 membered).

R^(102C) is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CN,—OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCCl₃, —OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —N₃,unsubstituted alkyl (e.g, C₁-C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄),unsubstituted heteroalkyl (e.g, 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to6, or 2 to 4 membered), unsubstituted cycloalkyl (e.g, C₃-C₈, C₃-C₆, orC₅-C₆), unsubstituted heterocycloalkyl (e.g, 3 to 8, 3 to 6, or 5 to 6membered), unsubstituted aryl (e.g, C₆-C₁₀, C₁₀, or phenyl), orunsubstituted heteroaryl (e.g, 5 to 10, 5 to 9, or 5 to 6 membered).

In embodiments, R^(102a) is independently hydrogen, halogen, —CCl₃,—CBr₃, —CF₃, —CI₃, —CHCl₂, —CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F,—CH₂I, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂,—NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH,—NHOH, —OCCl₃, —OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂,—OCH₂Cl, —OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, substituted (e.g,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted alkyl (e.g, C₁-C₈, C₁-C₆,C₁-C₄, or C₁-C₂), substituted (e.g, substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroalkyl (e.g, 2 to 8, 2 to 6, 4 to 6, 2 to 3, or 4 to5 membered), substituted (e.g, substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted cycloalkyl (e.g, C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆),substituted (e.g, substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheterocycloalkyl (e.g., 3 to 8, 3 to 6, 4 to 6, 4 to 5, or 5 to 6membered), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted aryl (e.g., C₆-C₁₀ or phenyl), or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroaryl (e.g., 5 to 10, 5to 9, or 5 to 6 membered).

In embodiments, a substituted R^(102a) (e.g., substituted alkyl,substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, substituted aryl, and/or substituted heteroaryl) issubstituted with at least one substituent group, size-limitedsubstituent group, or lower substituent group; wherein if thesubstituted R^(102a) is substituted with a plurality of groups selectedfrom substituent groups, size-limited substituent groups, and lowersubstituent groups; each substituent group, size-limited substituentgroup, and/or lower substituent group may optionally be different. Inembodiments, when R^(102a) is substituted, it is substituted with atleast one substituent group. In embodiments, when R^(102a) issubstituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when R^(102a) is substituted, it issubstituted with at least one lower substituent group. In embodiments,when R^(102a) is substituted, it is substituted with 1 to 10 substituentgroups. In embodiments, when R^(102a) is substituted, it is substitutedwith 1 to 10 size-limited substituent groups. In embodiments, whenR^(102a) is substituted, it is substituted with 1 to 10 lowersubstituent groups. In embodiments, when R^(102a) is substituted, it issubstituted with 1 to 5 substituent groups. In embodiments, whenR^(102a) is substituted, it is substituted with 1 to 5 size-limitedsubstituent groups. In embodiments, when R^(102a) is substituted, it issubstituted with 1 to 5 lower substituent groups. In embodiments, whenR^(102a) is substituted, it is substituted with a substituent group. Inembodiments, when R^(102a) is substituted, it is substituted with asize-limited substituent group. In embodiments, when R^(102a) issubstituted, it is substituted with a lower substituent group.

In embodiments, R^(102a) is independently hydrogen, halogen, —CCl₃,—CBr₃, —CF₃, —CI₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H,—SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H,—NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃, —OCF₃, —OCBr₃, —OCI₃, —OCHCl₂,—OCHBr₂, —OCHI₂, —OCHF₂, —N₃, unsubstituted alkyl (e.g., C₁-C₂₀,C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄), unsubstituted heteroalkyl (e.g., 2 to20, 8 to 20, 2 to 10, 2 to 8, 2 to 6, or 2 to 4 membered), unsubstitutedcycloalkyl (e.g., C₃-C₈, C₃-C₆, or C₅-C₆), unsubstitutedheterocycloalkyl (e.g., 3 to 8, 3 to 6, or 5 to 6 membered),unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, or phenyl), or unsubstitutedheteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered). In embodiments,R^(102a) is independently hydrogen or unsubstituted alkyl (e.g., C₁-C₂₀,C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄).

In embodiments, R^(102a) is independently unsubstituted alkyl (e.g.,C₁-C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄). In embodiments, R^(102a) isindependently unsubstituted C₁-C₆ alkyl. In embodiments, R^(102a) isindependently unsubstituted C₁-C₄ alkyl. In embodiments, R^(102a) isindependently unsubstituted methyl. In embodiments, R^(102a) isindependently unsubstituted tert-butyl. In embodiments, R^(102a) isindependently hydrogen.

In embodiments, R¹⁰² and R^(102a) are independently hydrogen orunsubstituted alkyl. In embodiments, R¹⁰² is unsubstituted C₁-C₄ alkyl.In embodiments, R^(102a) is hydrogen or unsubstituted methyl.

In embodiments, L¹⁰³ is independently a bond, —NH—, —O—, —C(O)—,—C(O)NH—, —NHC(O)—, —NHC(O)NH—, —C(O)O—, —OC(O)—, substituted orunsubstituted alkylene, substituted or unsubstituted heteroalkylene,substituted or unsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkyl ene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroaryl ene.

In embodiments, L¹⁰³ is a bond, —NH—, —S—, —O—, —C(O)—, —C(O)O—,—OC(O)—, —NHC(O)—, —C(O)NH—, —NHC(O)NH—, —NHC(NH)NH—, —C(S)—, —N═N—,—SS—, substituted or unsubstituted alkylene (e.g., C₁-C₂₀, C₁₀-C₂₀,C₁-C₈, C₁-C₆, or C₁-C₄), substituted or unsubstituted heteroalkylene(e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to 6, or 2 to 4 membered),substituted or unsubstituted cycloalkylene (e.g., C₃-C₈, C₃-C₆, orC₅-C₆), substituted or unsubstituted heterocycloalkyl ene (e.g., 3 to 8,3 to 6, or 5 to 6 membered), substituted or unsubstituted arylene (e.g.,C₆-C₁₀, C₁₀, or phenylene), or substituted or unsubstitutedheteroarylene (e.g., 5 to 10, 5 to 9, or 5 to 6 membered).

In embodiments, L¹⁰³ is a bond, —NH—, —NR¹⁰³—, —S—, —O—, —C(O)—,—C(O)O—, —OC(O)—, —NHC(O)—, —C(O)NH—, —NHC(O)NH—, —NHC(NH)NH—, —C(S)—,—N═N—, —SS—, R¹⁰³-substituted or unsubstituted alkylene (e.g., C₁-C₂₀,C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄), R¹⁰³-substituted or unsubstitutedheteroalkylene (e.g., 2 to 20 membered, 8 to 20 membered, 5 to 16membered, 2 to 10, 2 to 8, 2 to 6, or 2 to 4 membered), R¹⁰³-substitutedor unsubstituted cycloalkylene (e.g., C₃-C₈, C₃-C₆, or C₅-C₆),R¹⁰³-substituted or unsubstituted heterocycloalkylene (e.g., 3 to 8, 3to 6, or 5 to 6 membered), R¹⁰³-substituted or unsubstituted arylene(e.g., C₆-C₁₀, C₁₀, or phenylene), or R¹⁰³-substituted or unsubstitutedheteroarylene (e.g., 5 to 10, 5 to 9, or 5 to 6 membered). Inembodiments, L¹⁰³ is a bond. In embodiments, L¹⁰³ is —NH—. Inembodiments, L¹⁰³ is —NR¹⁰³—. In embodiments, L¹⁰³ is —S—. Inembodiments, L¹⁰³ is —O—. In embodiments, L¹⁰³ is —C(O)—. Inembodiments, L¹⁰³ is —C(O)O—. In embodiments, L¹⁰³ is —OC(O)—. Inembodiments, L¹⁰³ is —NHC(O)—. In embodiments, L¹⁰³ is —C(O)NH—. Inembodiments, L¹⁰³ is —NHC(O)NH—. In embodiments, L¹⁰³ is —NHC(NH)NH—. Inembodiments, L¹⁰³ is —C(S)—. In embodiments, L¹⁰³ is —N═N—. Inembodiments, L¹⁰³ is —SS—. In embodiments, L¹⁰³ is R¹⁰³-substituted orunsubstituted C₁-C₂₀ alkylene. In embodiments, L¹⁰³ is R¹⁰³-substitutedor unsubstituted 2 to 20 membered heteroalkylene. In embodiments, L¹⁰³is R¹⁰³-substituted or unsubstituted 5 to 16 membered heteroalkylene. Inembodiments, L¹⁰³ is R¹⁰³-substituted or unsubstituted 2 to 10 memberedheteroalkylene. In embodiments, L¹⁰³ is R¹⁰³-substituted orunsubstituted C₃-C₈ cycloalkylene. In embodiments, L¹⁰³ isR¹⁰³-substituted or unsubstituted 3 to 8 membered heterocycloalkylene.In embodiments, L¹⁰³ is R¹⁰³-substituted or unsubstituted C₆-C₁₀arylene. In embodiments, L¹⁰³ is R¹⁰³-substituted or unsubstituted 5 to10 membered heteroarylene.

In embodiments, L¹⁰³ is a bond, —NH—, —NR¹⁰³—, —S—, —O—, —C(O)—,—C(O)O—, —OC(O)—, —NHC(O)—, —C(O)NH—, —NHC(O)NH—, —NHC(NH)NH—, —C(S)—,—N═N—, —SS—, —CH(OH)—, or —C(CH₂)—. In embodiments, L¹⁰³ is a bond. Inembodiments, L¹⁰³ is —NH—. In embodiments, L¹⁰³ is —NR¹⁰³—. Inembodiments, L¹⁰³ is —S—. In embodiments, L¹⁰³ is —O—. In embodiments,L¹⁰³ is —C(O)—. In embodiments, L¹⁰³ is —C(O)O—. In embodiments, L¹⁰³ is—OC(O)—. In embodiments, L¹⁰³ is —NHC(O)—. In embodiments, L¹⁰³ is—C(O)NH—. In embodiments, L¹⁰³ is —NHC(O)NH—. In embodiments, L¹⁰³ is—NHC(NH)NH—. In embodiments, L¹⁰³ is —C(S)—. In embodiments, L¹⁰³ is—N═N—. In embodiments, L¹⁰³ is —SS—. In embodiments, L¹⁰³ is —CH(OH)—.In embodiments, L¹⁰³ is —C(CH₂)—.

In embodiments, L¹⁰³ is —(CH₂CH₂O)_(d)—. In embodiments, L¹⁰³ is—(CH₂O)_(d)—. In embodiments, L¹⁰³ is —(CH₂)_(d)—. In embodiments, L¹⁰³is —(CH₂)_(d)—NH—. In embodiments, L¹⁰³ is -(unsubstituted phenylene)-.In embodiments, L¹⁰³ is

In embodiments, L¹⁰³ is -(unsubstituted phenylene)-C(O)NH—. Inembodiments, L¹⁰³ is

In embodiments, L¹⁰³ is -(unsubstituted phenylene)-NHC(O)—. Inembodiments, L¹⁰³ is

The symbol d is an integer from 0 to 8. In embodiments, d is 3. Inembodiments, d is 1. In embodiments, d is 2. In embodiments, d is 0.

R¹⁰³ is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CN, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —N₃,R^(103A)-substituted or unsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀,C₁-C₈, C₁-C₆, or C₁-C₄), R^(103A)-substituted or unsubstitutedheteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to 6, or 2 to 4membered), R^(103A)-substituted or unsubstituted cycloalkyl (e.g.,C₃-C₈, C₃-C₆, or C₅-C₆), R^(103A)-substituted or unsubstitutedheterocycloalkyl (e.g., 3 to 8, 3 to 6, or 5 to 6 membered),R^(103A)-substituted or unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, orphenyl), or R^(103A)-substituted or unsubstituted heteroaryl (e.g., 5 to10, 5 to 9, or 5 to 6 membered).

In embodiments, R¹⁰³ is independently —NH₂. In embodiments, R¹⁰³ isindependently —OH. In embodiments, R¹⁰³ is independently halogen. Inembodiments, R¹⁰³ is independently —CN. In embodiments, R¹⁰³ isindependently oxo. In embodiments, R¹⁰³ is independently —CF₃. Inembodiments, R¹⁰³ is independently —COOH. In embodiments, R¹⁰³ isindependently —CONH₂. In embodiments, R¹⁰³ is independently —F. Inembodiments, R¹⁰³ is independently —Cl. In embodiments, R¹⁰³ isindependently —Br. In embodiments, R¹⁰³ is independently —I.

R^(103A) is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CN,—OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCCl₃, —OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —N₃,R^(103B)-substituted or unsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀,C₁-C₈, C₁-C₆, or C₁-C₄), R^(103B)-substituted or unsubstitutedheteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to 6, or 2 to 4membered), R^(103B)-substituted or unsubstituted cycloalkyl (e.g.,C₃-C₈, C₃-C₆, or C₅-C₆), R^(103B)-substituted or unsubstitutedheterocycloalkyl (e.g., 3 to 8, 3 to 6, or 5 to 6 membered),R^(103B)-substituted or unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, orphenyl), or R^(103B)-substituted or unsubstituted heteroaryl (e.g., 5 to10, 5 to 9, or 5 to 6 membered).

R^(103B) is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CN,—OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCCl₃, —OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —N₃,unsubstituted alkyl (e.g, C₁-C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄),unsubstituted heteroalkyl (e.g, 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to6, or 2 to 4 membered), unsubstituted cycloalkyl (e.g, C₃-C₈, C₃-C₆, orC₅-C₆), unsubstituted heterocycloalkyl (e.g, 3 to 8, 3 to 6, or 5 to 6membered), unsubstituted aryl (e.g, C₆-C₁₀, C₁₀, or phenyl), orunsubstituted heteroaryl (e.g, 5 to 10, 5 to 9, or 5 to 6 membered).

In embodiments, L¹⁰⁴ is independently a bond, —NH—, —O—, —C(O)—,—C(O)NH—, —NHC(O)—, —NHC(O)NH—, —C(O)O—, —OC(O)—, substituted orunsubstituted alkylene, substituted or unsubstituted heteroalkylene,substituted or unsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkyl ene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroaryl ene.

In embodiments, L¹⁰⁴ is a bond, —NH—, —S—, —O—, —C(O)—, —C(O)O—,—OC(O)—, —NHC(O)—, —C(O)NH—, —NHC(O)NH—, —NHC(NH)NH—, —C(S)—,substituted or unsubstituted alkylene (e.g, C₁-C₂₀, C₁₀-C₂₀, C₁-C₈,C₁-C₆, or C₁-C₄), substituted or unsubstituted heteroalkylene (e.g, 2 to20 membered, 8 to 20 membered, 5 to 16 membered, 2 to 10, 2 to 8, 2 to6, or 2 to 4 membered), substituted or unsubstituted cycloalkylene (e.g,C₃-C₈, C₃-C₆, or C₅-C₆), substituted or unsubstitutedheterocycloalkylene (e.g, 3 to 8, 3 to 6, or 5 to 6 membered),substituted or unsubstituted arylene (e.g, C₆-C₁₀, C₁₀, or phenylene),or substituted or unsubstituted heteroarylene (e.g, 5 to 10, 5 to 9, or5 to 6 membered).

In embodiments, L¹⁰⁴ is a bond, —NH—, —NR¹⁰⁴—, —S—, —O—, —C(O)—,—C(O)O—, —OC(O)—, —NHC(O)—, —C(O)NH—, —NHC(O)NH—, —NHC(NH)NH—, —C(S)—,R¹⁰⁴-substituted or unsubstituted alkylene (e.g, C₁-C₂₀, C₁₀-C₂₀, C₁-C₈,C₁-C₆, or C₁-C₄), R¹⁰⁴-substituted or unsubstituted heteroalkylene (e.g,2 to 20 membered, 8 to 20 membered, 5 to 16 membered, 2 to 10, 2 to 8, 2to 6, or 2 to 4 membered), R¹⁰⁴-substituted or unsubstitutedcycloalkylene (e.g., C₃-C₈, C₃-C₆, or C₅-C₆), R¹⁰⁴-substituted orunsubstituted heterocycloalkylene (e.g., 3 to 8, 3 to 6, or 5 to 6membered), R¹⁰⁴-substituted or unsubstituted arylene (e.g., C₆-C₁₀, C₁₀,or phenylene), or R¹⁰⁴-substituted or unsubstituted heteroarylene (e.g.,5 to 10, 5 to 9, or 5 to 6 membered). In embodiments, L¹⁰⁴ is a bond. Inembodiments, L¹⁰⁴ is —NH—. In embodiments, L¹⁰⁴ is —NR¹⁰⁴—. Inembodiments, L¹⁰⁴ is —S—. In embodiments, L¹⁰⁴ is —O—. In embodiments,L¹⁰⁴ is —C(O)—. In embodiments, L¹⁰⁴ is —C(O)O—. In embodiments, L¹⁰⁴ is—OC(O)—. In embodiments, L¹⁰⁴ is —NHC(O)—. In embodiments, L¹⁰⁴ is—C(O)NH—. In embodiments, L¹⁰⁴ is —NHC(O)NH—. In embodiments, L¹⁰⁴ is—NHC(NH)NH—. In embodiments, L¹⁰⁴ is —C(S)—. In embodiments, L¹⁰⁴ isR¹⁰⁴-substituted or unsubstituted C₁-C₂₀ alkylene. In embodiments, L¹⁰⁴is R¹⁰⁴-substituted or unsubstituted 2 to 20 membered heteroalkylene. Inembodiments, L¹⁰⁴ is R¹⁰⁴-substituted or unsubstituted 5 to 16 memberedheteroalkylene. In embodiments, L¹⁰⁴ is R¹⁰⁴-substituted orunsubstituted 2 to 10 membered heteroalkylene. In embodiments, L¹⁰⁴ isR¹⁰⁴-substituted or unsubstituted C₃-C₈ cycloalkylene. In embodiments,L¹⁰⁴ is R¹⁰⁴-substituted or unsubstituted 3 to 8 memberedheterocycloalkylene. In embodiments, L¹⁰⁴ is R¹⁰⁴-substituted orunsubstituted C₆-C₁₀ arylene. In embodiments, L¹⁰⁴ is R¹⁰⁴-substitutedor unsubstituted 5 to 10 membered heteroarylene. In embodiments, L¹⁰⁴ isR¹⁰⁴-substituted or unsubstituted phenylene.

In embodiments, L¹⁰⁴ is a bond, —NH—, —NR¹⁰⁴—, —S—, —O—, —C(O)—,—C(O)O—, —OC(O)—, —NHC(O)—, —C(O)NH—, —NHC(O)NH—, —NHC(NH)NH—, —C(S)—,—CH(OH)—, or —C(CH₂)—. In embodiments, L¹⁰⁴ is a bond. In embodiments,L¹⁰⁴ is —NH—. In embodiments, L¹⁰⁴ is —NR¹⁰⁴—. In embodiments, L¹⁰⁴ is—S—. In embodiments, L¹⁰⁴ is —O—. In embodiments, L¹⁰⁴ is —C(O)—. Inembodiments, L¹⁰⁴ is —C(O)O—. In embodiments, L¹⁰⁴ is —OC(O)—. Inembodiments, L¹⁰⁴ is —NHC(O)—. In embodiments, L¹⁰⁴ is —C(O)NH—. Inembodiments, L¹⁰⁴ is —NHC(O)NH—. In embodiments, L¹⁰⁴ is —NHC(NH)NH—. Inembodiments, L¹⁰⁴ is —C(S)—. In embodiments, L¹⁰⁴ is —CH(OH)—. Inembodiments, L¹⁰⁴ is —C(CH₂)—.

In embodiments, L¹⁰⁴ is —(CH₂CH₂O)_(e)—. In embodiments, L¹⁰⁴ is—(CH₂O)_(e)—. In embodiments, L¹⁰⁴ is —(CH₂)_(e)—. In embodiments, L¹⁰⁴is —(CH₂)_(e)—NH—. In embodiments, L¹⁰⁴ is -(unsubstituted phenylene)-.In embodiments, L¹⁰⁴ is

In embodiments, L¹⁰⁴ is -(unsubstituted phenylene)-C(O)NH—. Inembodiments, L¹⁰⁴ is

In embodiments, L¹⁰⁴ is -(unsubstituted phenylene)-NHC(O)—. Inembodiments, L¹⁰⁴ is

The symbol e is an integer from 0 to 8. In embodiments, e is 3. Inembodiments, e is 1. In embodiments, e is 2.

R¹⁰⁴ is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CN, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —N₃,R^(104A)-substituted or unsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀,C₁-C₈, C₁-C₆, or C₁-C₄), R^(104A)-substituted or unsubstitutedheteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to 6, or 2 to 4membered), R^(104A)-substituted or unsubstituted cycloalkyl (e.g.,C₃-C₈, C₃-C₆, or C₅-C₆), R^(104A)-substituted or unsubstitutedheterocycloalkyl (e.g., 3 to 8, 3 to 6, or 5 to 6 membered),R^(104A)-substituted or unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, orphenyl), or R^(104A)-substituted or unsubstituted heteroaryl (e.g., 5 to10, 5 to 9, or 5 to 6 membered).

In embodiments, R¹⁰⁴ is independently —NH₂. In embodiments, R¹⁰⁴ isindependently —OH. In embodiments, R¹⁰⁴ is independently halogen. Inembodiments, R¹⁰⁴ is independently —CN. In embodiments, R¹⁰⁴ isindependently oxo. In embodiments, R¹⁰⁴ is independently —CF₃. Inembodiments, R¹⁰⁴ is independently —COOH. In embodiments, R¹⁰⁴ isindependently —CONH₂. In embodiments, R¹⁰⁴ is independently —F. Inembodiments, R¹⁰⁴ is independently —Cl. In embodiments, R¹⁰⁴ isindependently —Br. In embodiments, R¹⁰⁴ is independently —I.

R^(104A) is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CN,—OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCCl₃, —OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —N₃,R^(104B)-substituted or unsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀,C₁-C₈, C₁-C₆, or C₁-C₄), R^(104B)-substituted or unsubstitutedheteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to 6, or 2 to 4membered), R^(104B)-substituted or unsubstituted cycloalkyl (e.g.,C₃-C₈, C₃-C₆, or C₅-C₆), R^(104B)-substituted or unsubstitutedheterocycloalkyl (e.g., 3 to 8, 3 to 6, or 5 to 6 membered),R^(104B)-substituted or unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, orphenyl), or R^(104B)-substituted or unsubstituted heteroaryl (e.g., 5 to10, 5 to 9, or 5 to 6 membered).

R^(104B) is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CN,—OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCCl₃, —OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —N₃,unsubstituted alkyl (e.g, C₁-C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄),unsubstituted heteroalkyl (e.g, 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to6, or 2 to 4 membered), unsubstituted cycloalkyl (e.g, C₃-C₈, C₃-C₆, orC₅-C₆), unsubstituted heterocycloalkyl (e.g, 3 to 8, 3 to 6, or 5 to 6membered), unsubstituted aryl (e.g, C₆-C₁₀, C₁₀, or phenyl), orunsubstituted heteroaryl (e.g, 5 to 10, 5 to 9, or 5 to 6 membered).

In embodiments, L¹⁰⁵ is independently a bond, —NH—, —O—, —C(O)—,—C(O)NH—, —NHC(O)—, —NHC(O)NH—, —C(O)O—, —OC(O)—, substituted orunsubstituted alkylene, substituted or unsubstituted heteroalkylene,substituted or unsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkyl ene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroaryl ene.

In embodiments, L¹⁰⁵ is a bond, —NH—, —S—, —O—, —C(O)—, —C(O)O—,—OC(O)—, —NHC(O)—, —C(O)NH—, —NHC(O)NH—, —NHC(NH)NH—, —C(S)—,substituted or unsubstituted alkylene (e.g, C₁-C₂₀, C₁₀-C₂₀, C₁-C₈,C₁-C₆, or C₁-C₄), substituted or unsubstituted heteroalkylene (e.g, 2 to20 membered, 8 to 20 membered, 5 to 16 membered, 2 to 10, 2 to 8, 2 to6, or 2 to 4 membered), substituted or unsubstituted cycloalkylene (e.g,C₃-C₈, C₃-C₆, or C₅-C₆), substituted or unsubstitutedheterocycloalkylene (e.g, 3 to 8, 3 to 6, or 5 to 6 membered),substituted or unsubstituted arylene (e.g, C₆-C₁₀, C₁₀, or phenylene),or substituted or unsubstituted heteroarylene (e.g, 5 to 10, 5 to 9, or5 to 6 membered).

In embodiments, L¹⁰⁵ is a bond, —NH—, —NR¹⁰⁵—, —S—, —O—, —C(O)—,—C(O)O—, —OC(O)—, —NHC(O)—, —C(O)NH—, —NHC(O)NH—, —NHC(NH)NH—, —C(S)—,R¹⁰⁵-substituted or unsubstituted alkylene (e.g, C₁-C₂₀, C₁₀-C₂₀, C₁-C₈,C₁-C₆, or C₁-C₄), R¹⁰⁵-substituted or unsubstituted heteroalkylene (e.g,2 to 20 membered, 8 to 20 membered, 5 to 16 membered, 2 to 10, 2 to 8, 2to 6, or 2 to 4 membered), R¹⁰⁵-substituted or unsubstitutedcycloalkylene (e.g., C₃-C₈, C₃-C₆, or C₅-C₆), R¹⁰⁵-substituted orunsubstituted heterocycloalkylene (e.g., 3 to 8, 3 to 6, or 5 to 6membered), R¹⁰⁵-substituted or unsubstituted arylene (e.g., C₆-C₁₀, C₁₀,or phenylene), or R¹⁰⁵-substituted or unsubstituted heteroarylene (e.g.,5 to 10, 5 to 9, or 5 to 6 membered). In embodiments, L¹⁰⁵ is a bond. Inembodiments, L¹⁰⁵ is —NH—. In embodiments, L¹⁰⁵ is —NR¹⁰⁵—. Inembodiments, L¹⁰⁵ is —S—. In embodiments, L¹⁰⁵ is —O—. In embodiments,L¹⁰⁵ is —C(O)—. In embodiments, L¹⁰⁵ is —C(O)O—. In embodiments, L¹⁰⁵ is—OC(O)—. In embodiments, L¹⁰⁵ is —NHC(O)—. In embodiments, L¹⁰⁵ is—C(O)NH—. In embodiments, L¹⁰⁵ is —NHC(O)NH—. In embodiments, L¹⁰⁵ is—NHC(NH)NH—. In embodiments, L¹⁰⁵ is —C(S)—. In embodiments, L¹⁰⁵ isR¹⁰⁵-substituted or unsubstituted C₁-C₂₀ alkylene. In embodiments, L¹⁰⁵is R¹⁰⁵-substituted or unsubstituted 2 to 20 membered heteroalkylene. Inembodiments, L¹⁰⁵ is R¹⁰⁵-substituted or unsubstituted 5 to 16 memberedheteroalkylene. In embodiments, L¹⁰⁵ is R¹⁰⁵-substituted orunsubstituted 2 to 10 membered heteroalkylene. In embodiments, L¹⁰⁵ isR¹⁰⁵-substituted or unsubstituted C₃-C₈ cycloalkylene. In embodiments,L¹⁰⁵ is R¹⁰⁵-substituted or unsubstituted 3 to 8 memberedheterocycloalkylene. In embodiments, L¹⁰⁵ is R¹⁰⁵-substituted orunsubstituted C₆-C₁₀ arylene. In embodiments, L¹⁰⁵ is R¹⁰⁵-substitutedor unsubstituted 5 to 10 membered heteroarylene.

In embodiments, L¹⁰⁵ is a bond, —NH—, —NR¹⁰⁵—, —S—, —O—, —C(O)—,—C(O)O—, —OC(O)—, —NHC(O)—, —C(O)NH—, —NHC(O)NH—, —NHC(NH)NH—, —C(S)—,—CH(OH)—, or —C(CH₂)—. In embodiments, L¹⁰⁵ is a bond. In embodiments,L¹⁰⁵ is —NH—. In embodiments, L¹⁰⁵ is —NR¹⁰⁵—. In embodiments, L¹⁰⁵ is—S—. In embodiments, L¹⁰⁵ is —O—. In embodiments, L¹⁰⁵ is —C(O)—. Inembodiments, L¹⁰⁵ is —C(O)O—. In embodiments, L¹⁰⁵ is —OC(O)—. Inembodiments, L¹⁰⁵ is —NHC(O)—. In embodiments, L¹⁰⁵ is —C(O)NH—. Inembodiments, L¹⁰⁵ is —NHC(O)NH—. In embodiments, L¹⁰⁵ is —NHC(NH)NH—. Inembodiments, L¹⁰⁵ is —C(S)—. In embodiments, L¹⁰⁵ is —CH(OH)—. Inembodiments, L¹⁰⁵ is —C(CH₂)—.

In embodiments, L¹⁰⁵ is —(CH₂CH₂O)_(f)—. In embodiments, L¹⁰⁵ is—(CH₂O)_(f)—. In embodiments, L¹⁰⁵ is —(CH₂)_(f)—. In embodiments, L¹⁰⁵is —(CH₂)_(f)—NH—. In embodiments, L¹⁰⁵ is —C(O)NH(CH₂)_(f)—NH—. Inembodiments, L¹⁰⁵ is —(CH₂CH₂O)_(f)—(CH₂)_(g)—NH—. In embodiments, L¹⁰⁵is —(CH₂)_(g)—. In embodiments, L¹⁰⁵ is —(CH₂)_(g)—NH—. In embodiments,L¹⁰⁵ is —NHC(O)—(CH₂)_(f)—NH—. In embodiments, L¹⁰⁵ is—NHC(O)—(CH₂)_(f)—NH—. In embodiments, L¹⁰⁵ is—NHC(O)—(CH₂CH₂O)_(f)—(CH₂)_(g)—NH—. In embodiments, L¹⁰⁵ is—NHC(O)—(CH₂)_(g-). In embodiments, L¹⁰⁵ is —NHC(O)—(CH₂)_(g)—NH—. Inembodiments, L¹⁰⁵ is —C(O)NH(CH₂)_(f)—NH—. In embodiments, L¹⁰⁵ is—C(O)NH—(CH₂CH₂O)_(f)—(CH₂)_(g)—NH—. In embodiments, L¹⁰⁵ is—C(O)NH—(CH₂)_(g-). In embodiments, L¹⁰⁵ is —C(O)NH—(CH₂)_(g)—NH—. Thesymbol f is an integer from 0 to 8. In embodiments, f is 3. Inembodiments, f is 1. In embodiments, f is 2. In embodiments, f is 0. Thesymbol g is an integer from 0 to 8. In embodiments, g is 3. Inembodiments, g is 1. In embodiments, g is 2. In embodiments, g is 0.

R¹⁰⁵ is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CN, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —N₃,R^(105A)-substituted or unsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀,C₁-C₈, C₁-C₆, or C₁-C₄), R^(105A)-substituted or unsubstitutedheteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to 6, or 2 to 4membered), R^(105A)-substituted or unsubstituted cycloalkyl (e.g.,C₃-C₈, C₃-C₆, or C₅-C₆), R^(105A)-substituted or unsubstitutedheterocycloalkyl (e.g., 3 to 8, 3 to 6, or 5 to 6 membered),R^(105A)-substituted or unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, orphenyl), or R^(105A)-substituted or unsubstituted heteroaryl (e.g., 5 to10, 5 to 9, or 5 to 6 membered).

In embodiments, R¹⁰⁵ is independently -MB. In embodiments, R¹⁰⁵ isindependently —OH. In embodiments, R¹⁰⁵ is independently halogen. Inembodiments, R¹⁰⁵ is independently —CN. In embodiments, R¹⁰⁵ isindependently oxo. In embodiments, R¹⁰⁵ is independently —CF₃. Inembodiments, R¹⁰⁵ is independently —COOH. In embodiments, R¹⁰⁵ isindependently —CONH₂. In embodiments, R¹⁰⁵ is independently —F. Inembodiments, R¹⁰⁵ is independently —Cl. In embodiments, R¹⁰⁵ isindependently —Br. In embodiments, R¹⁰⁵ is independently —I.

R^(105A) is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CN,—OH, -MB, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCCl₃, —OCF₃, —OCBr₃, —OCB, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —N₃,R^(105B)-substituted or unsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀,C₁-C₈, C₁-C₆, or C₁-C₄), R^(105B)-substituted or unsubstitutedheteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to 6, or 2 to 4membered), R^(105B)-substituted or unsubstituted cycloalkyl (e.g.,C₃-C₈, C₃-C₆, or C₅-C₆), R^(105B)-substituted or unsubstitutedheterocycloalkyl (e.g., 3 to 8, 3 to 6, or 5 to 6 membered),R^(105B)-substituted or unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, orphenyl), or R^(105B)-substituted or unsubstituted heteroaryl (e.g., 5 to10, 5 to 9, or 5 to 6 membered).

R^(105B) is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CN,—OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCCl₃, —OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —N₃,unsubstituted alkyl (e.g, C₁-C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄),unsubstituted heteroalkyl (e.g, 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to6, or 2 to 4 membered), unsubstituted cycloalkyl (e.g, C₃-C₈, C₃-C₆, orC₅-C₆), unsubstituted heterocycloalkyl (e.g, 3 to 8, 3 to 6, or 5 to 6membered), unsubstituted aryl (e.g, C₆-C₁₀, C₁₀, or phenyl), orunsubstituted heteroaryl (e.g, 5 to 10, 5 to 9, or 5 to 6 membered).

In embodiments, L¹⁰¹, L¹⁰³, L¹⁰⁴, and L¹⁰⁵ are independently a bond,substituted or unsubstituted alkylene, substituted or unsubstitutedheteroalkylene, substituted or unsubstituted cycloalkylene, substitutedor unsubstituted heterocycloalkylene, substituted or unsubstitutedarylene, or substituted or unsubstituted heteroarylene.

In embodiments, L¹⁰¹ is independently a substituted or unsubstitutedC₁-C₄ alkylene or substituted or unsubstituted 8 to 20 memberedheteroalkylene; L¹⁰³ is independently a bond or substituted orunsubstituted 2 to 10 membered heteroalkylene; L¹⁰⁴ is independently abond, substituted or unsubstituted 4 to 18 membered heteroalkylene, orsubstituted or unsubstituted phenylene; L¹⁰⁵ is independently bond orsubstituted or unsubstituted 4 to 18 membered heteroalkylene.

In embodiments, L¹⁰¹, L¹⁰³, and L¹⁰⁵ are independently a bond, —NH—,—O—, —C(O)—, —C(O)NH—, —NHC(O)—, —NHC(O)NH—, —C(O)O—, —OC(O)—,substituted or unsubstituted alkylene, substituted or unsubstitutedheteroalkylene, substituted or unsubstituted cycloalkylene, substitutedor unsubstituted heterocycloalkylene, substituted or unsubstitutedarylene, or substituted or unsubstituted heteroarylene. In embodiments,L¹⁰⁴ is unsubstituted phenylene.

In embodiments, L¹⁰¹ is independently a substituted or unsubstitutedC₁-C₄ alkylene or substituted or unsubstituted 8 to 20 memberedheteroalkylene. In embodiments, L¹⁰³ is independently a bond orsubstituted or unsubstituted 2 to 10 membered heteroalkylene. Inembodiments, L¹⁰⁴ is independently an unsubstituted phenylene. Inembodiments, L¹⁰⁵ is independently bond or substituted or unsubstituted4 to 18 membered heteroalkylene.

In embodiments, L¹⁰¹ is independently a substituted or unsubstitutedC₁-C₄ alkylene or substituted or unsubstituted 8 to 20 memberedheteroalkylene; L¹⁰³ is independently a bond or substituted orunsubstituted 2 to 10 membered heteroalkylene; L¹⁰⁴ is independently anunsubstituted phenylene; L¹⁰⁵ is independently bond or substituted orunsubstituted 4 to 18 membered heteroalkylene; and R¹⁰² is unsubstitutedC₁-C₄ alkyl; and R^(102a) is hydrogen or unsubstituted methyl.

In embodiments, -(L¹⁰¹)-OC(SSR¹⁰²)(R^(102a))-(L¹⁰³)-(L¹⁰⁴)-(L¹⁰⁵)- is

R¹⁰² is as described herein, including in embodiments.

In embodiments, -(L¹⁰¹)-OC(SSR¹⁰²)(R^(102a))-(L¹⁰³)-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-OC(SSR¹⁰²)(R^(102a))-(L¹⁰³)-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-OC(SSR¹⁰²)(R^(102a))-(L¹⁰³)-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-OC(SSR¹⁰²)(R^(102a))-(L¹⁰³)-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-OC(SSR¹⁰²)(R^(102a))-(L¹⁰³)-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-OC(SSR¹⁰²)(R^(102a))-(L¹⁰³)-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-OC(SSR¹⁰²)(R^(102a))-(L¹⁰³)-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-OC(SSR¹⁰²)(R^(102a))-(L¹⁰³)-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-OC(SSR¹⁰²)(R^(102a))-(L¹⁰³)-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-OC(SSR¹⁰²)(R^(102a))-(L¹⁰³)-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-OC(SSR¹⁰²)(R^(102a))-(L¹⁰³)-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-OC(SSR¹⁰²)(R^(102a))-(L¹⁰³)-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-OC(SSR¹⁰²)(R^(102a))-(L¹⁰³)-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-OC(SSR¹⁰²)(R^(102a))-(L¹⁰³)-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-OC(SSR¹⁰²)(R^(102a))-(L¹⁰³)-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-OC(SSR¹⁰²)(R^(102a))-(L¹⁰³)-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-OC(SSR¹⁰²)(R^(102a))-(L¹⁰³)-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-OC(SSR¹⁰²)(R^(102a))-(L¹⁰³)-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-OC(SSR¹⁰²)(R^(102a))-(L¹⁰³)-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-OC(SSR¹⁰²)(R^(102a))-(L¹⁰³)-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-OC(SSR¹⁰²)(R^(102a))-(L¹⁰³)-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-OC(SSR¹⁰²)(R^(102a))-(L¹⁰³)-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-OC(SSR¹⁰²)(R^(102a))-(L¹⁰³)-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-OC(SSR¹⁰²)(R^(102a))-(L¹⁰³)-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-OC(SSR¹⁰²)(R^(102a))-(L¹⁰³)-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-OC(SSR¹⁰²)(R^(102a))-(L¹⁰³)-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-OC(SCN)(R^(102a))-(L¹⁰³)-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-OC(SCN)(R^(102a))-(L¹⁰³)-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-OC(SCN)(R^(102a))-(L¹⁰³)-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-OC(SCN)(R^(102a))-(L¹⁰³)-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-OC(SCN)(R^(102a))-(L¹⁰³)-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-OC(SCN)(R^(102a))-(L¹⁰³)-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-OC(SCN)(R^(102a))-(L¹⁰³)-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-OC(SCN)(R^(102a))-(L¹⁰³)-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-OC(SCN)(R^(102a))-(L¹⁰³)-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, L¹⁰¹ is —CCCH₂—NHC(O)—O—(CH₂)_(c)—. In embodiments, L¹⁰¹is —CCCH₂—NHC(O)—NH—(CH₂)_(c)—. In embodiments, L¹⁰¹ is—CCCH₂—NHC(O)—(CH₂)_(c)—. The symbol c is an integer from 0 to 8. Inembodiments, c is 0. In embodiments, c is 1. In embodiments, c is 2. Inembodiments, c is 3. In embodiments, c is 4. In embodiments, L¹⁰¹ is—CCCH₂—NHC(O)—O—(CH₂)₄—. In embodiments, L¹⁰¹ is—CCCH₂—NHC(O)—NH—(CH₂)₄—. In embodiments, L¹⁰¹ is —CCCH₂—NHC(O)—(CH₂)₂₋.In embodiments, L¹⁰¹ is —CCCH₂—NHC(O)—O—(CH₂)_(c)—OCH(R¹⁰²)—. Inembodiments, L¹⁰¹ is —CCCH₂—NHC(O)—NH—(CH₂)_(c)—OCH(R¹⁰²)—.

In embodiments, L¹⁰² is a bond. In embodiments, L¹⁰² is —OCH(R¹⁰²)—. Inembodiments, L¹⁰² is —OCH(CH₃)—. In embodiments, L¹⁰² is

In embodiments, L¹⁰² is

In embodiments, L¹⁰³ is —SS—. In embodiments, R¹⁰² is —CH₃.

In embodiments, L¹⁰⁴ is a bond. In embodiments, L¹⁰⁴ is —(CH₂CH₂O)_(e)—.In embodiments, L¹⁰⁴ is —(C(CH₃)₂)—(CH₂CH₂O)_(e)—. In embodiments, L¹⁰⁴is —(CH₂)_(e)—. In embodiments, L¹⁰⁴ is —(CH₂)_(e)—NH—. In embodiments,L¹⁰⁴ is —(C(CH₃)₂)—(CH₂CH₂O)—. In embodiments, L¹⁰⁴ is —(CH₂)—NH—. Inembodiments, L¹⁰⁴ is —(CH₂)₂—NH—. In embodiments, L¹⁰⁴ is-(unsubstituted phenylene)-. In embodiments, L¹⁰⁴ is

The symbol e is an integer from 0 to 8. In embodiments, e is 3. Inembodiments, e is 1. In embodiments, e is 2. In embodiments, e is 0. Inembodiments, L¹⁰⁴ is -(unsubstituted phenylene)-CH₂C(O)NH—. Inembodiments, L¹⁰⁴ is —(C(CH₃)₂)—(CH₂CH₂O)—C(O)NH—.

In embodiments, L¹⁰⁵ is a bond. In embodiments, L¹⁰⁵ is —(CH₂)_(f)—NH—.In embodiments, L¹⁰⁵ is —(CH₂)₂—NH—. In embodiments, L¹⁰⁵ is—C(O)NH(CH₂)_(f)—NH—. In embodiments, L¹⁰⁵ is—(CH₂CH₂O)_(f)—(CH₂)_(g)—NH—. In embodiments, L¹⁰⁵ is—CH₂—C(O)NH—(CH₂CH₂O)_(f)—(CH₂)_(g)—NH—. In embodiments, L¹⁰⁵ is—CH₂—C(O)NH—(CH₂CH₂O)₃—(CH₂)₂—NH—. In embodiments, L¹⁰⁵ is—C(O)NH—(CH₂CH₂O)_(f)—(CH₂)_(g)—NH—. In embodiments, L¹⁰⁵ is—C(O)NH—(CH₂CH₂O)₂—(CH₂)₂—NH—. The symbol f is an integer from 0 to 8.In embodiments, f is 3. In embodiments, f is 1. In embodiments, f is 2.In embodiments, f is 0. The symbol g is an integer from 0 to 8. Inembodiments, g is 3. In embodiments, g is 1. In embodiments, g is 2. Inembodiments, g is 0.

In embodiments, L¹⁰⁰ is -(L¹⁰¹)-(L¹⁰²)-SS-(L¹⁰⁴)-(L¹⁰⁵)-. L¹⁰¹, L¹⁰²,L¹⁰⁴, and L¹⁰⁵ are as described herein. In embodiments, L¹⁰⁰ is-(L¹⁰¹)-OCH(R¹⁰²)—SS-(L¹⁰⁴)-(L¹⁰⁵)-. L¹⁰¹, L¹⁰⁴, and L¹⁰⁵ are asdescribed herein.

In embodiments, -(L¹⁰¹)-(L¹⁰²)-SS-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-(L¹⁰²)-SS-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-(L¹⁰²)-SS-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-(L¹⁰²)-SS-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-(L¹⁰²)-SS-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-(L¹⁰²)-SS-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-(L¹⁰²)-SS-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-(L¹⁰²)-SS-(L¹⁰⁴)-(L¹⁰⁵)- is

R¹⁰² is as described herein, including in embodiments.

In embodiments, -(L¹⁰¹)-(L¹⁰²)-SS-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-(L¹⁰²)-SS-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-(L¹⁰²)-SS-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-(L¹⁰²)-SS-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-(L¹⁰²)-SS-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-(L¹⁰²)-SS-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-(L¹⁰²)-SS-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-(L¹⁰²)-SS-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-(L¹⁰²)-SS-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-(L¹⁰²)-SS-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-(L¹⁰²)-SS-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-(L¹⁰²)-SS-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-(L¹⁰²)-SS-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-(L¹⁰²)-SS-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-(L¹⁰²)-SS-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, -(L¹⁰¹)-(L¹⁰²)-SS-(L¹⁰⁴)-(L¹⁰⁵)- is

In embodiments, L¹⁰¹ is

In embodiments, L¹⁰¹ is

In embodiments, L¹⁰¹ is —CCCH₂—. In embodiments, L¹⁰¹ is

In embodiments, L¹⁰¹ is

In embodiments, L¹⁰¹ is

In embodiments, L¹⁰³ is

In embodiments, L¹⁰³ is

In embodiments, L¹⁰³ is

In embodiments, L¹⁰³ is

In embodiments, L¹⁰³ is

In embodiments, L¹⁰³ is a bond.

In embodiments, L¹⁰⁴ is

In embodiments, L¹⁰⁴ is

In embodiments, L¹⁰⁴ is

In embodiments, L¹⁰⁴ is

In embodiments, L¹⁰⁴ is

In embodiments, L¹⁰⁴ is a bond.

In embodiments, L¹⁰⁵ is

In embodiments, L¹⁰⁵ is

In embodiments, L¹⁰⁵ is

In embodiments, L¹⁰⁵ is

In embodiments, L¹⁰⁵ is

In embodiments, L¹⁰⁵ is a bond.

In embodiments, L¹⁰³-L¹⁰⁴-L¹⁰⁵- is

In embodiments, L¹⁰³-L¹⁰⁴-L¹⁰⁵- is

In embodiments, L¹⁰³-L¹⁰⁴-L¹⁰⁵- is

In embodiments, L¹⁰³-L¹⁰⁴-L¹⁰⁵- is

In embodiments, L¹⁰³-L¹⁰⁴-L¹⁰⁵- is

In embodiments, L¹⁰⁰ is

R¹⁰⁰ is as described herein, including in embodiments.In embodiments, B is

In embodiments, L¹⁰⁰ is

R¹⁰² is as described herein, including in embodiments.In embodiments, B is

In embodiments, L¹⁰⁰ is

In embodiments, B is

In embodiments, L¹⁰⁰ is

In embodiments, B is

In embodiments, L¹⁰⁰ is

In embodiments, B is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

R¹⁰⁰ is as described herein, including in embodiments.

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

R¹⁰² is as described herein, including in embodiments.

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

wherein R⁹, L¹⁰⁴, L¹⁰⁵, and R¹⁰² are as described herein. Inembodiments, L¹⁰⁰ is

wherein R⁹, L¹⁰⁴, L¹⁰⁵, and R¹⁰² are as described herein.

In embodiments, L¹⁰⁰ is

wherein R⁹, L¹⁰⁴, L¹⁰⁵, and R¹⁰² are as described herein. Inembodiments, L¹⁰⁰ is

wherein R⁹, L¹⁰⁴, L¹⁰⁵, and R¹⁰² are as described herein.

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, L¹⁰⁰ is

In embodiments, R⁴ is a detectable moiety. In embodiments, R⁴ is afluorescent dye moiety. In embodiments, R⁴ is a detectable moietydescribed herein (e.g., Table 1). In embodiments, R⁴ is a detectablemoiety described in Table 1.

TABLE 1 Detectable moieties to be used in selected embodiments.Nucleoside/nucleotide λmax abbreviation Dye name (nm) dC Atto 532 532 dCAtto Rho 6G 535 dC R6G 534 dC Tet 521 dT Atto Rho 11 572 dT Atto 565 564dT Alexa Fluor 568 578 dT dTamra 578 dA Alexa Fluor 647 650 dA Atto 647N644 dA Janelia Fluor 646 646 dG Alexa Fluor 680 682 dG Alexa Fluor 700696 dG CF680R 680

In embodiments, R⁴ is

In embodiments, the compound has the formula:

wherein R¹, R⁷, R⁸, B¹, and R² are as described herein. In embodiments,the compound has the formula:

wherein R¹, R⁷, R⁸, B¹, and R² are as described herein.

In embodiments, the compound has the formula:

wherein R¹, B¹, and R² are as described herein. In embodiments, thecompound has the formula:

wherein R¹, B¹, and R² are as described herein. In embodiments, thecompound has the formula:

wherein R¹, B¹, and R² are as described herein. In embodiments, thecompound has the formula:

wherein R¹, B¹, and R² are as described herein.

In embodiments, the compound has the formula:

wherein B¹, R⁷, and R⁸ are as described herein, including embodiments.In embodiments, the compound has the formula:

wherein B¹ and R⁷ are as described herein, including embodiments.

In embodiments, the compound has the formula:

wherein B¹, and R⁷ are as described herein, including embodiments. Inembodiments, R⁷ is a substituted or unsubstituted aryl or substituted orunsubstituted heteroaryl. In embodiments, R⁷ is a substituted orunsubstituted heteroaryl wherein the atom bonded to the thiocarbonylcarbon is not a heteroatom (e.g., nitrogen).

In embodiments, the compound has the formula:

wherein B, R⁷, L¹⁰⁰, and R⁴ are as described herein, including inembodiments.

In embodiments, the compound has the formula:

wherein B, L¹⁰⁰, and R⁴ are as described herein, including inembodiments. In embodiments, the compound has the formula:

wherein z7, B, L¹⁰⁰, R^(7A), and R⁴ are as described herein, includingin embodiments. In embodiments, the compound has the formula:

wherein B, L¹⁰⁰, R^(7A), and R⁴ are as described herein, including inembodiments. In embodiments, the compound has the formula:

wherein B, L¹⁰⁰, R^(7A), and R⁴ are as described herein, including inembodiments. In embodiments, the compound has the formula:

wherein B, L¹⁰⁰, and R⁴ are as described herein, including inembodiments. In embodiments, the compound has the formula:

wherein B, L¹⁰⁰, and R⁴ are as described herein, including inembodiments. In embodiments, the compound has the formula:

wherein B, L¹⁰⁰, and R⁴ are as described herein, including inembodiments.

In embodiments, the compound has the formula:

R⁷, L¹⁰⁰, and R⁴ are as described herein, including in embodiments. Inembodiments, L¹⁰⁰ is a cleavable linker. In embodiments, R⁴ is afluorescent dye moiety. In embodiments, R⁷ is a substituted orunsubstituted aryl or substituted or unsubstituted heteroaryl. Inembodiments, R⁷ is a substituted or unsubstituted cycloalkyl orsubstituted or unsubstituted heterocycloalkyl. In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, the compound has the formula:

L¹⁰⁰ and R⁴ are as described herein, including in embodiments. Inembodiments, L¹⁰⁰ is a cleavable linker. In embodiments, R⁴ is afluorescent dye moiety.

In embodiments, the compound has the formula:

L¹⁰⁰ and R⁴ are as described herein, including in embodiments. Inembodiments, L¹⁰⁰ is a cleavable linker. In embodiments, R⁴ is afluorescent dye moiety.

In embodiments, the compound has the formula:

L¹⁰⁰ and R⁴ are as described herein, including in embodiments. Inembodiments, L¹⁰⁰ is a cleavable linker. In embodiments, R⁴ is afluorescent dye moiety.

In embodiments, the compound has the formula:

R¹, B, R⁷, R⁸, L¹⁰¹, L¹⁰², L¹⁰⁴, L¹⁰⁵, and R⁴ are as described herein,including in embodiments. In embodiments, the compound has the formula:

R¹, B, L¹⁰¹, L¹⁰², L¹⁰⁴, L¹⁰⁵, and R⁴ are as described herein, includingin embodiments. In embodiments, the compound has the formula:

R¹, B, L¹⁰¹, L¹⁰², L¹⁰⁴, L¹⁰⁵, and R⁴ are as described herein, includingin embodiments. In embodiments, the compound has the formula:

R¹, B, L¹⁰¹, L¹⁰², L¹⁰⁴, L¹⁰⁵, and R⁴ are as described herein, includingin embodiments. In embodiments, the compound has the formula:

R¹, B, L¹⁰¹, L¹⁰², L¹⁰⁴, L¹⁰⁵, and R⁴ are as described herein, includingin embodiments.

In embodiments, the compound has the formula:

R¹, B, R⁷, R⁸, L¹⁰¹, L¹⁰⁴, L¹⁰⁵, and R⁴ are as described herein,including in embodiments. In embodiments, the compound has the formula:

R¹, B, R⁷, R⁸, L¹⁰¹, L¹⁰⁴, L¹⁰⁵, and R⁴ are as described herein,including in embodiments. In embodiments, the compound has the formula:

R¹, B, R⁷, R⁸, R¹⁰², L¹⁰¹, L¹⁰³, L¹⁰⁴, L¹⁰⁵, and R⁴ are as describedherein, including in embodiments. In embodiments, the compound has theformula:

R¹, B, R⁷, R⁸, R¹⁰², L¹⁰¹, L¹⁰⁴, L¹⁰⁵, and R⁴ are as described herein,including in embodiments. In embodiments, the compound has the formula:

R¹, B, R⁷, R⁸, L¹⁰¹, L¹⁰⁴, L¹⁰⁵, and R⁴ are as described herein,including in embodiments. In embodiments, the compound has the formula:

R¹, B, R⁷, R⁸, R¹⁰², L¹⁰¹, L¹⁰³, L¹⁰⁴, L¹⁰⁵, and R⁴ are as describedherein, including in embodiments. In embodiments, the compound has theformula:

R¹, B, R⁷, R⁸, R¹⁰², L¹⁰¹, L¹⁰⁴, L¹⁰⁵, and R⁴ are as described herein,including in embodiments. In embodiments, L¹⁰¹ is

In embodiments, R⁸ is an unsubstituted C₁-C₂ alkyl.

In embodiments, the compound has the formula:

wherein B, R⁷, R⁸, R⁹, L¹⁰⁴, L¹⁰⁵, and R⁴ are as described herein,including in embodiments. In embodiments, the compound has the formula:

wherein B, R⁷, R⁸, R⁹, L¹⁰⁴, L¹⁰⁵, and R⁴ are as described herein,including in embodiments. In embodiments, the compound has the formula:

wherein B, R⁷, R⁸, R¹⁰², L¹⁰³, L¹⁰⁴, L¹⁰⁵, and R⁴ are as describedherein, including in embodiments. In embodiments, the compound has theformula:

R⁸, R¹⁰², L¹⁰⁴, L¹⁰⁵, and R⁴ are as described herein, including inembodiments. In embodiments, R⁸ is an unsubstituted C₁-C₂ alkyl.

In embodiments, the compound has the formula:

wherein L¹⁰⁰ is a cleavable linker. In embodiments, L¹⁰⁰ is

wherein R⁹, L¹⁰⁴, L¹⁰⁵, and R¹⁰² are as described herein.

In embodiments, the compound is not a compound described in WO2017/079498.

In an aspect is provided a nucleic acid polymerase complex, wherein thenucleic acid polymerase is bound (e.g., non-covalently bound) to acompound described herein, including embodiments. In embodiments, thenucleic acid polymerase is bound to a primer.

In embodiments, the nucleic acid polymerase is a Taq polymerase,Therminator γ, 9° N polymerase (exo-), Therminator II, Therminator III,or Therminator IX. In embodiments, the nucleic acid polymerase isTherminator γ. In embodiments, the nucleic acid polymerase is 9° Npolymerase (exo-). In embodiments, the nucleic acid polymerase isTherminator II. In embodiments, the nucleic acid polymerase isTherminator III. In embodiments, the nucleic acid polymerase isTherminator IX. In embodiments, the nucleic acid polymerase is a Taqpolymerase. In embodiments, the nucleic acid polymerase is a nucleicacid polymerase. In embodiments, the nucleic acid polymerase is 9° N andmutants thereof. In embodiments, the nucleic acid polymerase is Phi29and mutants thereof. In embodiments, the DNA polymerase is a modifiedarchaeal DNA polymerase. In embodiments, the polymerase is a reversetranscriptase. In embodiments, the polymerase is a mutant P. abyssipolymerase (e.g., such as a mutant P. abyssi polymerase described in WO2018/148723 or WO 2020/056044).

In embodiments, the 3′ moiety of a compound described herein ischemically cleaved faster than a control nucleotide. In embodiments, the3′ moiety of a compound described herein is chemically cleaved fasterthan a nucleotide with a 3′-OCH₂SSCH₃ moiety under identical cleavageconditions (e.g., same reaction time, same reaction temperature, and/orthe same reducing agent). In embodiments, the 3′ moiety of a compounddescribed herein is chemically cleaved faster than a nucleotide with a3′-OCH₂N₃ moiety under identical cleavage conditions (e.g., samereaction time, same reaction temperature, and/or the same reducingagent). In embodiments, a compound of formula I, II, or III (e.g., in anaspect or embodiment) is chemically cleaved faster than an identicalcompound wherein the 3′-O-reversible terminator portion is

for Formula I, II, and III respectively is replaced with a 3′-OCH₂SSCH₃(e.g., under identical cleavage conditions). In embodiments, a compoundof formula I (e.g., in an aspect or embodiment) is chemically cleavedfaster than an identical compound wherein the 3′-OCH(phenyl)SSCH₃ isreplaced with a 3′-OCH₂SSCH₃ (e.g., under identical cleavageconditions).

In embodiments, chemical cleavage of a compound described herein (e.g.,in an aspect or embodiment) is at least 1.1-fold (e.g., 1.1, 1.2, 1.3,1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40,50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000,10000, or 100000-fold) faster than chemical cleavage of an identicalcompound wherein the 3′-OCH(R⁷)SSCH₃ is replaced with a 3′-OCH₂SSCH₃ or3′-OCH₂N₃ (e.g., under identical cleavage conditions). In embodiments,chemical cleavage of a compound described herein (e.g., in an aspect orembodiment) is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300,400, 500, 600, 700, 800, 900, 1000, 10000, or about 100000-fold fasterthan chemical cleavage of an identical compound wherein the3′-OCH(R⁷)SSCH₃ is replaced with a 3′-OCH₂SSCH₃ or 3′-OCH₂N₃ (e.g.,under identical cleavage conditions). In embodiments, chemical cleavageof a compound described herein (e.g., in an aspect or embodiment) is1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800,900, 1000, 10000, or 100,000-fold faster than chemical cleavage of anidentical compound wherein the 3′-OCH(phenyl)SSCH₃ is replaced with a3′-OCH₂SSCH₃ or 3′-OCH₂N₃ (e.g., under identical cleavage conditions).In embodiments, chemical cleavage is cleavage of the SS bond (e.g., in a3′ moiety). In embodiments, chemical cleavage is release of a 3′ moietyfrom a nucleotide, nucleoside, or residue (e.g., from being bound to the3′ carbon of the sugar) to leave a 3′-OH on the nucleotide, nucleoside,or residue (e.g., attached to 3′ carbon of the sugar). In embodiments,chemical cleavage is cleavage of the SS bond in a 3′ moiety and releaseof a 3′ moiety from a nucleotide, nucleoside, or residue (e.g., frombeing bound to the 3′ carbon of the sugar) to leave a 3′-OH on thenucleotide, nucleoside, or residue (e.g., attached to 3′ carbon of thesugar).

In embodiments, chemical cleavage of a compound (e.g., cleavage of the3′ moiety of a compound described herein or cleavage of an SS bond in a3′ moiety of a compound described herein) described herein (e.g., in anaspect or embodiment) includes contacting the compound with a reducingagent (e.g., tris(hydroxypropyl)phosphine (THPP),tris-(2-carboxyethyl)phosphine (TCEP), tris(hydroxymethyl)phosphine(THMP), or tris(hydroxyethyl)phosphine (THEP), DTT, dithiobutylamine(DTBA)). In embodiments, chemical cleavage of a compound (e.g., cleavageof the 3′ moiety of a compound described herein or cleavage of an SSbond in a 3′ moiety of a compound described herein) described herein(e.g., in an aspect or embodiment) includes contacting the compound withTHPP (e.g., about 10 mM THPP, or at least 1 mM THPP). In embodiments,chemical cleavage of a compound (e.g., cleavage of the 3′ moiety of acompound described herein or cleavage of an SS bond in a 3′ moiety of acompound described herein) described herein (e.g., in an aspect orembodiment) is performed at less than about 65° C. In embodiments,chemical cleavage of a compound (e.g., cleavage of the 3′ moiety of acompound described herein or cleavage of an SS bond in a 3′ moiety of acompound described herein) described herein (e.g., in an aspect orembodiment) is performed at less than 65° C. In embodiments, chemicalcleavage of a compound (e.g., cleavage of the 3′ moiety of a compounddescribed herein or cleavage of an SS bond in a 3′ moiety of a compounddescribed herein) described herein (e.g., in an aspect or embodiment) isperformed at about 45-65° C. In embodiments, chemical cleavage of acompound (e.g., cleavage of the 3′ moiety of a compound described hereinor cleavage of an SS bond in a 3′ moiety of a compound described herein)described herein (e.g., in an aspect or embodiment) is performed at45-65° C. In embodiments, chemical cleavage of a compound (e.g.,cleavage of the 3′ moiety of a compound described herein or cleavage ofan SS bond in a 3′ moiety of a compound described herein) describedherein (e.g., in an aspect or embodiment) is performed at 45° C., 46°C., 47° C., 48° C., 49° C., 50° C., 51° C., 52° C., 53° C., 54° C., 55°C., 56° C., 57° C., 58° C., 59° C., 60° C., 61° C., 62° C., 63° C., 64°C., or 65° C. In embodiments, chemical cleavage of a compound (e.g.,cleavage of the 3′ moiety of a compound described herein or cleavage ofan SS bond in a 3′ moiety of a compound described herein) describedherein (e.g., in an aspect or embodiment) is performed at about 55° C.In embodiments, chemical cleavage of a compound (e.g., cleavage of the3′ moiety of a compound described herein or cleavage of an SS bond in a3′ moiety of a compound described herein) described herein (e.g., in anaspect or embodiment) is performed at a temperature of at least 55° C.In embodiments, chemical cleavage of a compound (e.g., cleavage of the3′ moiety of a compound described herein or cleavage of an SS bond in a3′ moiety of a compound described herein) described herein (e.g., in anaspect or embodiment) is performed at about pH 9.5. In embodiments,chemical cleavage of a compound (e.g., cleavage of the 3′ moiety of acompound described herein or cleavage of an SS bond in a 3′ moiety of acompound described herein) described herein (e.g., in an aspect orembodiment) is performed at about pH 9.5. In embodiments, chemicalcleavage of a compound (e.g., cleavage of the 3′ moiety of a compounddescribed herein or cleavage of an SS bond in a 3′ moiety of a compounddescribed herein) described herein (e.g., in an aspect or embodiment) isperformed at pH 9.5. In embodiments, chemical cleavage of a compound(e.g., cleavage of the 3′ moiety of a compound described herein orcleavage of an SS bond in a 3′ moiety of a compound described herein)described herein (e.g., in an aspect or embodiment) is performed using0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 mM of THPP. Inembodiments, the chemical cleavage is performed using less than 1.0 mMTHPP. In embodiments, the chemical cleavage is performed using about 1.0mM THPP. In embodiments, the chemical cleavage is performed using about0.05 to about 1.0 mM THPP. In embodiments, the chemical cleavage isperformed using about 1.0 to about 5.0 mM THPP. In embodiments, thechemical cleavage is performed using about 10 mM THPP. In embodiments,the chemical cleavage is performed using 1.0 mM THPP. In embodiments,the chemical cleavage is performed using about 0.05 to 1.0 mM THPP. Inembodiments, the chemical cleavage is performed using 1.0 to about 5.0mM THPP. In embodiments, the chemical cleavage is performed using 10 mMTHPP.

In embodiments, the compounds described herein provide superiorstability in solution during storage, or reagent handling duringsequencing applications, compared to the same compounds that have 3′-OHreversible terminating groups disclosed in the prior art, such as forexample the 3′-O-azidomethyl reversible terminator.

In an aspect is provided a kit. Some embodiments disclosed herein relateto kits including a labeled nucleoside or nucleotide (e.g., a compoundas described herein) including a linker between the fluorophore and thenucleoside or nucleotide, wherein the linker is a linker as describedherein. In embodiments, the kit includes a compound described herein. Inembodiments, the kit includes a plurality of compounds described herein.In embodiments, the kit includes labeled nucleotides includingdifferently labeled nucleotides (e.g., compounds described herein). Inembodiments, the kit further includes instructions for use thereof. Inembodiments, kits described herein include a polymerase. In embodiments,the polymerase is a DNA polymerase. In embodiments, the DNA polymeraseis a thermophilic nucleic acid polymerase. In embodiments, the DNApolymerase is a modified archaeal DNA polymerase. In embodiments, thekit includes a sequencing solution. In embodiments, the sequencingsolution include labeled nucleotides including differently labelednucleotides, wherein the label (or lack thereof) identifies the type ofnucleotide. For example, each adenine nucleotide, or analog thereof; athymine nucleotide; a cytosine nucleotide, or analog thereof; and aguanine nucleotide, or analog thereof may be labeled with a differentfluorescent label.

In embodiments, the sequencing solution includes a buffer solution.Typically, the buffered solutions contemplated herein are made from aweak acid and its conjugate base or a weak base and its conjugate acid.For example, sodium acetate and acetic acid are buffer agents that canbe used to form an acetate buffer. Other examples of buffer agents thatcan be used to make buffered solutions include, but are not limited to,Tris, Tricine, HEPES, TES, MOPS, MOPSO and PIPES. Additionally, otherbuffer agents that can be used in enzyme reactions, hybridizationreactions, and detection reactions are well known in the art. Inembodiments, the buffered solution can include Tris. With respect to theembodiments described herein, the pH of the buffered solution can bemodulated to permit any of the described reactions. In some embodiments,the buffered solution can have a pH greater than pH 7.0, greater than pH7.5, greater than pH 8.0, greater than pH 8.5, greater than pH 9.0,greater than pH 9.5, greater than pH 10, greater than pH 10.5, greaterthan pH 11.0, or greater than pH 11.5. In other embodiments, thebuffered solution can have a pH ranging, for example, from about pH 6 toabout pH 9, from about pH 8 to about pH 10, or from about pH 7 to aboutpH 9. In embodiments, the buffered solution can comprise one or moredivalent cations. Examples of divalent cations can include, but are notlimited to, Mg²⁺, Mn²⁺, Zn²⁺, and Ca²⁺. In embodiments, the bufferedsolution can contain one or more divalent cations at a concentrationsufficient to permit hybridization of a nucleic acid. In someembodiments, a concentration can be more than about 1 μM, more thanabout 2 μM, more than about 5 μM, more than about 10 μM, more than about25 μM, more than about 50 μM, more than about 75 μM, more than about 100μM, more than about 200 μM, more than about 300 μM, more than about 400μM, more than about 500 μM, more than about 750 μM, more than about 1mM, more than about 2 mM, more than about 5 mM, more than about 10 mM,more than about 20 mM, more than about 30 mM, more than about 40 mM,more than about 50 mM, more than about 60 mM, more than about 70 mM,more than about 80 mM, more than about 90 mM, more than about 100 mM,more than about 150 mM, more than about 200 mM, more than about 250 mM,more than about 300 mM, more than about 350 mM, more than about 400 mM,more than about 450 mM, more than about 500 mM, more than about 550 mM,more than about 600 mM, more than about 650 mM, more than about 700 mM,more than about 750 mM, more than about 800 mM, more than about 850 mM,more than about 900 mM, more than about 950 mM or more than about 1 M.

III. Methods of Use

In an aspect is provided a method for sequencing a nucleic acid,including (i) incorporating in series with a nucleic acid polymerase,within a reaction vessel, one of four different compounds into a primerto create an extension strand, wherein the primer is hybridized to thenucleic acid and wherein each of the four different compounds includes aunique detectable label; (ii) detecting the unique detectable label ofeach incorporated compound, so as to thereby identify each incorporatedcompound in the extension strand, thereby sequencing the nucleic acid;wherein each of the four different compounds is independently a compoundas described herein, including embodiments. In embodiments, the compoundincludes at least one of the following: cytosine or a derivativethereof, guanine or a derivative thereof, adenine or a derivativethereof, thymine or a derivative thereof, uracil or a derivativethereof, hypoxanthine or a derivative thereof, xanthine or a derivativethereof, 7-methyl guanine or a derivative thereof, 5,6-dihydrouracil ora derivative thereof, 5-methylcytosine or a derivative thereof, and5-hydroxymethylcytosine or a derivative thereof. In embodiments, thecompound includes at least one of the following: cytosine or aderivative thereof, guanine or a derivative thereof, adenine or aderivative thereof, thymine or a derivative thereof, and uracil or aderivative thereof. In embodiments, the compound includes at least oneof the following: cytosine or a derivative thereof, guanine or aderivative thereof, adenine or a derivative thereof, and thymine or aderivative thereof. In embodiments, the compound includes at least oneof the following: cytosine or a derivative thereof, guanine or aderivative thereof, adenine or a derivative thereof, and uracil or aderivative thereof. In embodiments, the method further includes, afterincorporating, contacting the compound with a cleaving agent. Inembodiments, the method includes incorporating a first nucleotideincluding a 3′-O-reversible terminator (e.g.,

and a first detectable label; detecting the first detectable label; andremoving the 3′-O-reversible terminator from the first nucleotide togenerate a nucleotide including a 3′-OH. In embodiments, the methodincludes generating one or more sequencing reads.

In embodiments, the nucleic acid to be sequenced is DNA or RNA, or ahybrid molecule comprised of deoxynucleotides and ribonucleotides. Inembodiments, the nucleic acid to be sequenced is attached to a solidsubstrate via any suitable linkage method known in the art, e.g., usingcovalent linkage. In embodiments, the nucleic acid is attached directlyto a solid substrate. In embodiments, the surface of the solid supportincludes a polymer that provides the attachment points for the nucleicacid.

In embodiments, the nucleic acid is within a cluster. The terms“cluster” and “colony” are used interchangeably throughout thisapplication and refer to a discrete site on a solid support comprised ofa plurality of immobilized nucleic acid strands. The term “clusteredarray” refers to an array formed from such clusters or colonies. In thiscontext the term “array” is not to be understood as requiring an orderedarrangement of clusters. The term “array” is used in accordance with itsordinary meaning in the art, and refers to a population of differentmolecules that are attached to one or more solid-phase substrates suchthat the different molecules can be differentiated from each otheraccording to their relative location. An array can include differentmolecules that are each located at different addressable features on asolid-phase substrate. The molecules of the array can be nucleic acidprimers, nucleic acid probes, nucleic acid templates or nucleic acidenzymes such as polymerases or ligases. Arrays useful in the inventioncan have densities that ranges from about 2 different features to manymillions, billions or higher. The density of an array can be from 2 toas many as a billion or more different features per square cm. Forexample an array can have at least about 100 features/cm², at leastabout 1,000 features/cm², at least about 10,000 features/cm², at leastabout 100,000 features/cm², at least about 10,000,000 features/cm², atleast about 100,000,000 features/cm², at least about 1,000,000,000features/cm², at least about 2,000,000,000 features/cm² or higher. Inembodiments, the arrays have features at any of a variety of densitiesincluding, for example, at least about 10 features/cm², 100features/cm², 500 features/cm², 1,000 features/cm², 5,000 features/cm²,10,000 features/cm², 50,000 features/cm², 100,000 features/cm²,1,000,000 features/cm², 5,000,000 features/cm², or higher.

In an aspect is provided a method of incorporating a compound into aprimer, the method including combining a polymerase, a primer hybridizedto nucleic acid template and the compound within a reaction vessel andallowing the polymerase to incorporate the compound into the primerthereby forming an extended primer, wherein the compound is a compoundas described herein, including embodiments. In embodiments,incorporating a compound into a primer refers to the 5′ phosphatejoining in phosphodiester linkage to the 3′—OH group of a second(modified or unmodified) nucleotide, which may itself form part of alonger polynucleotide chain.

In embodiments, the method further including, after each of theincorporating steps, adding to the reaction vessel four differentunlabeled compounds (e.g., nucleotide analogues). In embodiments, theunlabeled compounds have the formula:

wherein R¹, R², and B¹ are as described herein, and R³ is apolymerase-compatible cleavable moiety or reversible terminator (e.g.,

as described herein. In embodiments, B¹ is a monovalent nucleobase(e.g., is

In embodiments, each of the four different unlabeled compounds (e.g.,nucleotide analogues) are of the structure as described herein,including embodiments, wherein in the first of the four differentunlabeled compounds, B¹ is a thymidine or uridine hybridizing base; inthe second of the four different unlabeled compounds, B¹ is an adenosinehybridizing base; in the third of the four different unlabeledcompounds, B¹ is a guanosine hybridizing base; and in the fourth of thefour different unlabeled compounds, B¹ is a cytosine hybridizing base.

In embodiments, the nucleic acid polymerase is a Taq polymerase,Therminator γ, 9° N polymerase (exo-), Therminator II, Therminator III,or Therminator IX. In embodiments, the nucleic acid polymerase isTherminator γ. In embodiments, the nucleic acid polymerase is 9° Npolymerase (exo-). In embodiments, the nucleic acid polymerase isTherminator II. In embodiments, the nucleic acid polymerase isTherminator III. In embodiments, the nucleic acid polymerase isTherminator IX. In embodiments, the nucleic acid polymerase is a Taqpolymerase. In embodiments, the nucleic acid polymerase is a nucleicacid polymerase. In embodiments, the nucleic acid polymerase is 9° N andmutants thereof. In embodiments, the nucleic acid polymerase is Phi29and mutants thereof. In embodiments, the DNA polymerase is a modifiedarchaeal DNA polymerase. In embodiments, the polymerase is a reversetranscriptase. In embodiments, the polymerase is a mutant P. abyssipolymerase (e.g., such as a mutant P. abyssi polymerase described in WO2018/148723 or WO 2020/056044, both of which are incorporated byreference herein). In embodiments, the polymerase is DNA polymerase, aterminal deoxynucleotidyl transferase, or a reverse transcriptase. Inembodiments, the enzyme is a DNA polymerase, such as DNA polymerase 812(Pol 812) or DNA polymerase 1901 (Pol 1901), e.g., a polymerasedescribed in US 2020/0131484, and US 2020/0181587, both of which areincorporated by reference herein.

In embodiments, the method includes simultaneously sequencing aplurality of different nucleic acids, including: a) extending aplurality of primer DNA strands hybridized to template DNAs, each ofwhich includes one of the primer DNA strands, by incorporating a labelednucleotide (i.e., a compound as described herein) in the presence of anenzyme; and b) identifying each labeled nucleotide, so as tosimultaneously sequence the plurality of different nucleic acids. Inembodiments, the labeled nucleotide is a compound described herein.

In an aspect is provided a method of determining the sequence of atarget single-stranded polynucleotide. In embodiments, the methodincludes incorporating a compound as described herein, (e.g., a compoundof Formula I, Formula II, or Formula III) into an oligonucleotide strandcomplementary to at least a portion of the target polynucleotide strand;and detecting the identity of the compound incorporated into theoligonucleotide strand. In embodiments, the compound includes a3′-O-polymerase-compatible cleavable moiety as described herein and adetectable label. In embodiments, the method further includes chemicallyremoving the detectable label and the 3′-O-polymerase-compatiblecleavable moiety from the compound incorporated into the oligonucleotidestrand. In embodiments, the 3′-O-polymerase-compatible cleavable moietyand the detectable label of the incorporated compound are removed priorto introducing the next complementary compound. In embodiments, the3′-O-polymerase-compatible cleavable moiety and the detectable label areremoved in a single step of chemical reaction. In embodiments, thesequential incorporation described herein is performed at least 50times, at least 100 times, at least 150 times, at least 200 times, atleast 250 times, at least 300 times, at least 350 times, at least 400times, at least 450 times, or at least 500 times. In embodiments, thesequential incorporation is performed 80 to 200 times. In embodiments,the sequential incorporation is performed 100 to 200 times. Inembodiments, the sequential incorporation is performed 120 to 250 times.

In embodiments, the method further including, after the incorporating,cleaving the linker (e.g., L¹⁰⁰ or-(L¹⁰¹)-OC(SR¹⁰⁰)(R^(102a))-(L¹⁰³)-(L¹⁰⁴)-(L¹⁰⁵)-) with a cleavingreagent (e.g., tris(hydroxypropyl)phosphine (THPP), dithiobutylamine(DTBA), or DTT). In embodiments, the cleaving reagent is in a buffer. Inembodiments, the buffer includes an acetate buffer,3-(N-morpholino)propanesulfonic acid (MOPS) buffer,N-(2-Acetamido)-2-aminoethanesulfonic acid (ACES) buffer,phosphate-buffered saline (PBS) buffer,4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer,N-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid(AMPSO) buffer, borate buffer (e.g., borate buffered saline, sodiumborate buffer, boric acid buffer), 2-Amino-2-methyl-1,3-propanediol(AMPD) buffer, N-cyclohexyl-2-hydroxyl-3-aminopropanesulfonic acid(CAPSO) buffer, 2-Amino-2-methyl-1-propanol (AMP) buffer,4-(Cyclohexylamino)-1-butanesulfonic acid (CABS) buffer, glycine-NaOHbuffer, N-Cyclohexyl-2-aminoethanesulfonic acid (CHES) buffer,tris(hydroxymethyl)aminomethane (Tris) buffer, or aN-cyclohexyl-3-aminopropanesulfonic acid (CAPS) buffer. In embodiments,the buffer is a borate buffer. In embodiments, the buffer is a CHESbuffer.

In embodiments, the method further including, after the incorporating,cleaving the linker (e.g., L¹⁰⁰ or-(L¹⁰¹)-OC(SR¹⁰⁰)(R^(102a))-(L¹⁰³)-(L¹⁰⁴)-(L¹⁰⁵)-) with a cleavingreagent (e.g., a water-soluble phosphine, such astris(hydroxypropyl)phosphine (THPP)). In embodiments, the cleavingreagent is a reducing agent. In embodiments, the cleaving agent is aphosphine containing agent. In embodiments, the cleaving agent is athiol containing agent. In embodiments, the cleaving agent isdi-mercaptopropane sulfonate (DMPS). In embodiments, the cleaving agentis aqueous sodium sulfide (Na₂S). In embodiments, the cleaving reagentis Tris-(2-carboxyethyl)phosphines trisodium salt (TCEP),tris(hydroxypropyl)phosphine (THPP), guanidine, urea, cysteine,2-mercaptoethylamine, or dithiothreitol (DTT). In embodiments, thecleaving reagent is an acid, base, oxidizing agent, reducing agent,Pd(0), tris-(2-carboxyethyl)phosphine, dilute nitrous acid, fluoride,tris(3-hydroxypropyl)phosphine), sodium dithionite (Na₂S₂O₄), orhydrazine (N₂H₄). In embodiments, the method includes contacting thecompound (e.g., a compound described herein) with a reducing agent. Inembodiments, the method further including, after the incorporating,cleaving the linker at about 55° C. In embodiments, the method furtherincluding, after the incorporating, cleaving the linker at about 45° C.to about 60° C. In embodiments, the method further including, after theincorporating, cleaving the linker at about 55° C. to about 80° C. Inembodiments, the method further including, after the incorporating,cleaving the linker at about 60° C. to about 70° C. In embodiments, themethod further including, after the incorporating, cleaving the linkerat about 50° C. to about 60° C. In embodiments, the method furtherincluding, after the incorporating, cleaving the linker at about 65° C.to about 75° C. In embodiments, the method further including, after theincorporating, cleaving the linker at about 65° C. In embodiments, themethod further including, after the incorporating, cleaving the linkerat about 55° C. In embodiments, the method further including, after theincorporating, cleaving the linker at about 55° C., 56° C., 57° C., 58°C., 59° C., 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., 66° C., 67°C., 68° C., 69° C., 70° C., 71° C., 72° C., 73° C., 74° C., 75° C., 76°C., 77° C., 78° C., 79° C., or about 80° C. In embodiments, the methodfurther including, after the incorporating, cleaving the linker at a pHat about 8.0 to 11.0. In embodiments, the pH is 9.0 to 11.0. Inembodiments, the pH is 9.5. In embodiments, the pH is 10.0. Inembodiments, the pH is 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, or 11.0. Inembodiments, the pH is from 9.0 to 11.0, and the temperature is about60° C. to about 70° C. In embodiments, the pH is from 9.0 to 11.0, andthe temperature is about 50° C. to about 60° C.

In embodiments, the cleaving reagent cleaves both the linker (e.g.,L¹⁰⁰) and the polymerase-compatible cleavable moiety (e.g., the3′-O-reversible terminator

for Formula I, II, and III respectively) simultaneously.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

EXAMPLES Example 1. Novel Modified Nucleotides

In the context of nucleic acid sequencing, the use of nucleotidesbearing a 3′ reversible terminator (RT) (also referred to herein as apolymerase-compatible cleavable moiety) allows successive nucleotides tobe incorporated into a polynucleotide chain in a controlled manner. TheDNA template for a sequencing reaction will typically comprise adouble-stranded region having a free 3′ hydroxyl group which serves as aprimer or initiation point for the addition of further nucleotides inthe sequencing reaction. The region of the DNA template to be sequencedwill overhang this free 3′ hydroxyl group on the complementary strand.The primer bearing the free 3′ hydroxyl group may be added as a separatecomponent (e.g., a short oligonucleotide) which hybridizes to a regionof the template to be sequenced. Following the addition of a singlenucleotide to the DNA template, the presence of the 3′ reversibleterminator prevents incorporation of a further nucleotide into thepolynucleotide chain. While the addition of subsequent nucleotides isprevented, the identity of the incorporated nucleotide is detected(e.g., exciting a unique detectable label that is linked to theincorporated nucleotide). The reversible terminator is then removed (andoptionally the cleavable linker is removed simultaneously), leaving afree 3′ hydroxyl group for addition of the next nucleotide. Thesequencing cycle can then continue with the incorporation of the nextblocked, labeled nucleotide.

Sequencing by synthesis of nucleic acids ideally requires the controlled(i.e., one at a time), yet rapid, incorporation of the correctcomplementary nucleotide opposite the oligonucleotide being sequenced.This allows for accurate sequencing by adding nucleotides in multiplecycles as each nucleotide residue is sequenced one at a time, thuspreventing an uncontrolled series of incorporations occurring.Nucleotides bearing a 3′ RT have been described in the literature, seefor example U.S. Pat. No. 6,664,079 or Ju J. et al. (2006) Proc NatlAcad. Sci USA 103(52): 19635-19640, however despite the recent advancesonly a few solutions have been presented, most of which cause otherproblems, including inefficient or incomplete incorporation by thepolymerase, inefficient or incomplete cleavage of the removable group,or harsh conditions needed for the cleaving step causing spuriousproblems with the remainder of the assay and/or fidelity of the targetsequence.

There are many limitations on types of reversible terminators that canbe added onto a nucleotide and still be suitable. The reversibleterminator should prevent additional nucleotide molecules from beingadded to the polynucleotide while simultaneously being easily removablefrom the sugar moiety without causing damage to the polynucleotide orsequencing enzyme (e.g., DNA polymerase or reverse transcriptase). Idealreversible terminators therefore possess long term stability, can beefficiently incorporated by the sequencing enzyme, can prevent secondaryor further nucleotide incorporation, and have the ability to be removedunder mild conditions that do not cause damage to any sequencingcomponent (e.g., nucleotides, primers, enzymes, polymers, etc.)preferably under aqueous conditions. Developing a truly reversible setof nucleotide RTs and cleavable linkers that are stable and cleaverapidly has been a goal for many years.

An important property of a reversible terminator on a nucleotide is thatit can be rapidly cleaved under conditions that do not adversely affectthe DNA. Removal of a disulfide containing reversible terminator to formthe 3′-OH requires the formation of a thiol, followed by conversion to ahydroxide (see scheme 1), via a tandem nucleophilic fragmentationreaction. FIG. 1A describes a simplified schematic identifying apotential transition state.

The stability of the resultant thioaldehyde influences the cleavage rateof the thioacetal. For example, an RT having the structure

(referred to as a methylene disulfide, or RT #1), wherein the oxygen isattached to the 3′ position of the deoxyribose, results in the formationof thioformaldehyde, a notoriously unstable molecule which rapidlyoligomerizes to 1,3,5-trithiane. Thioformaldehydes are highly reactiveand inherently unstable species due to the lack of steric and resonancestabilization afforded to the sp2 carbon by the hydrogens. In accordancewith the Hammond Postulate, the transition state is geometrically moresimilar to the thioaldehyde for this particular reaction (see forexample March's Advanced Organic Chemistry, 6th Ed., Wiley, 2007,Michael B. Smith and Jerry March, Chapter 6 Methods of determiningmechanisms, page 308). Conceptualizing the thioaldehyde with allavailable resonance geometries (see FIG. 1B) suggests a stable ylidestructure that is geometrically similar to the resultant thioaldehydewill be more thermodynamically favored. Therefore, increasing theresonance stabilization to the sp2 carbon by including aresonance-stabilizing moiety (e.g., a cyclic moiety, such as an aromaticor heteroaromatic moiety) involves only a small reorganization of themolecular structures and thus permits faster cleavage. This concept isfurther illustrated in FIG. 1B and supported vide infra. For example, anRT that includes a methyl substituent on the methylene carbon having thestructure

(RT #2) increases the cleavage rate approximately 10-fold relative to RT#1. In contrast, substituents incapable of forming a stabilized ylide(e.g., substituents described in US 2016/0002721, such as —CH₂F or—CHF₂) are likely not stabilizing. Additionally, merely extending thealkyl chain

(RT #2a) did not improve the cleavage rate relative to RT #2. Withoutwishing to be bound by theory, aliphatic thioaldehydes (e.g., such asthe thioaldehyde produced when cleaving RT #2 or RT #2a) may polymerizeand their isolation may be problematic, suggesting substituents whichfurther stabilize the resultant thioaldehyde (i.e., have a greaternumber of resonant structures) results in an increased cleavage rate.Aromatic thioaldehydes are more stable (see Moldoveanu, S. Chapter10—Pyrolysis of Aldehydes and Ketones, Pyrolysis of Organic Molecules(Second Edition), Elsevier, 2019, Pages 391-418), therefore the cleavagerate of disulfide-containing reversible terminators (i.e., nucleotideshaving formula I, II, or III as described herein comprising thereversible terminator at the 3′O-position,

that produce aromatic thioaldehydes increases relative to a methylenedisulfide. Thus, the modified nucleotides as described herein are stableand rapidly cleaved under mild conditions.

Example 2. Chemical Synthesis of Modified Nucleotides

Included in Scheme 2 is a generalized overview for synthesizing amodified nucleotide as described herein. Introducing theresonance-stabilizing moiety (e.g., cyclic moiety such as an aromatic orheteroaromatic moiety) in the reversible terminator is achieved byintroducing a functionalized acetal. The acetal may be formed via analdehyde conversion reaction using mild conditions, as depicted inScheme 5, and reported in Grabowski et al Org. Biomol. Chem., 2018, 16,3114.

Alternatively, the starting nucleoside may include a protected propargylamine off the base, as shown in Scheme 4. Nucleosides containing aprotected propargyl amine off the base may be used as the inputnucleoside in Scheme 4, following a similar protocol to produce areversible terminator containing nucleotide with a protected propargylamine off the base. The protecting group is then removed by exposing thenucleoside containing a protected propargyl amine off the base toconcentrated ammonium hydroxide.

Included in Scheme 7 is a generalized overview for synthesizing amodified nucleotide of Formula III, wherein R⁷ is as described herein.

presence of copper, e.g., copper sulfate or

Example 3. Incorporation of Modified Nucleotides

A modified nucleotide (e.g., the nucleotide compounds described herein)should be capable of being rapidly incorporated by a DNA polymerase.Naturally occurring DNA polymerases are typically not capable ofincorporating nucleotides modified with reversible terminator at the 3′position on the ribose of the nucleotide. As known in the art, a numberof thermophilic polymerases have been engineered to enable theincorporation of nucleotides modified with 3′ terminators. For example,the thermophilic polymerase is a mutant P. abyssi polymerase (e.g., suchas a mutant P. abyssi polymerase described in WO 2018/148723 or WO2020/056044, both incorporated herein by reference for any use).

The data presented within Table 2 shows the half-time for incorporationof RT #2, RT #3, RT #45, RT #22, and RT #26, by a modified thermophilicpolymerase. The reaction was carried out in a buffered solution at pH8.5, with nucleotides at 200 nM concentration, 4 mM Mg, at a temperatureof 55° C. The polymerase was pre-bound to the primed DNA template. Allterminated nucleotides were efficiently incorporated by the modified DNApolymerase. Surprisingly, the incorporation of RT #2, RT #3, RT #26, RT#22 are comparable, despite the latter compounds (i.e., RT #3, RT #22,and RT #26) bearing relatively bulkier substituents. For the largernaphthalenyl substituent, RT #45, the polymerase takes nearly twice aslong to incorporate the nucleotide. The average incorporation half timeis reported in Table 2.

TABLE 2 Incorporation halftime for a variety of compounds describedherein, keeping the nucleobase, enzyme, buffer, and temperature the samefor all experiments. The symbol R⁸ is indicative of the R⁸ substituentas provided in Formula II. ^(†)RT#3 is averaged over all fournucleotides (dA, dT, dC, and dG). Incorporation Half time (s) RT #2m 8.8± 3 R⁸ = unsubstituted methyl RT #3m  6.3 ± 3^(†) R⁸ = unsubstitutedmethyl RT #3e 5.3 ± 3 R⁸ = unsubstituted ethyl RT #3np 10.5 ± 3  R⁸ =unsubstituted n-propyl RT #3ip 15.1 ± 3  R⁸ = unsubstituted isopropyl RT#45e; 25.3 ± 3  R⁸ = unsubstituted ethyl RT #26e 6.3 ± 3 R⁸ =unsubstituted ethyl RT #13e 23.3 ± 3  R⁸ = unsubstituted ethyl

Example 4. Cleavage Kinetics

An important property of a reversible terminator on a nucleotide is thatit can be rapidly cleaved under conditions that do not adversely affectthe DNA. FIGS. 2A-2B reports the cleavage halftime rates for threedifferent reversible terminated nucleotides. RT #1 is

wherein the oxygen is attached to the 3′ position of the deoxyribose; RT#2 is the methyl-substituted methylene, e.g., having the formula

wherein the oxygen is attached to the 3′ position of the deoxyribose;and RT #3 is

wherein the oxygen is attached to the 3′ position of the deoxyribose. Tocalculate the cleavage half time, a dTTP nucleotide with each 3′ moietywas incorporated into a growing DNA strand immobilized on a solidsupport. Excess nucleotides were washed away. Next, a cleavage solutioncontaining 1 mM THPP as a reducing agent was introduced for controlledperiods of time. The cleavage reaction was carried out at 55° C., in abuffered solution at 9.5 pH. The cleavage reaction for RT #1 was slow at55° C., as shown in FIG. 2A, so to make a meaningful comparison to theother RT moieties, the temperature for only RT #1 was increased to 65°C., as shown in FIG. 2B. As observed in FIGS. 2A-2C, there is a drasticimprovement in the cleavage kinetics (i.e., a reduction in halftime) ofRT #3 compared to RT #1 and RT #2. While the cleavage of the disulfidebond (reversible terminator) is rapid in both cases, the subsequenthydrolysis reaction that removes the residual portion of the 3′ moietyis much faster with the new 3′ moieties of Formula I, II, and III.Modifying the reaction conditions (e.g., elevating the temperature to65° C., increasing the pH, increasing the amount or concentration of thereducing agent) results in faster cleavage.

The kinetics of the disulfide cleavage are not significantly affected bythe terminal alkyl group. As indicated in Table 2, the incorporationkinetics are more sensitive to the terminal alkyl group (i.e., R⁸ inFormula II). Data presented within Table 3 show that when R⁷ is methyland R⁸ is methyl or ethyl, the kinetics are relatively invariant.Similarly, when R⁷ is unsubstituted phenyl and R⁸ is unsubstitutedmethyl, unsubstituted ethyl, or unsubstituted propyl, the cleavagekinetics are comparable. Advantageously, the cleavage kinetics for thecompounds described herein are surprisingly 10× faster than controlcompounds (e.g., when R⁷ and R⁸ are methyl). Each sequencing cycle isdirectly correlated to the cleavage kinetics; faster cleavage results ina faster sequencing cycle. Sequencing a 150 bp template polynucleotidewhich typically takes about 18 hours of sequencing time, assuming a 7minute sequencing cycle, can now be accomplished in about 5-8 hoursusing the nucleotides described herein.

TABLE 3 Cleavage kinetics measurements varying R⁷ and R⁸ for dTTPnucleotides. Cleavage conditions include measurements of disulfidecleavage using 0.1 mM THPP at 55° C.

Cleavage R⁷ R⁸ halftime (s)

21.2 ± 3

20.3 ± 3

21.1 ± 3

 1.6 ± 3

 2.8 ± 3

 2.9 ± 3

 0.9 ± 3

 2.7 ± 3

 2.0 ± 3

 3.5 ± 3

Example 5. Chemical Stability

A modified nucleotide is of no use in sequencing if it is not chemicalstable during storage and use. Any degradation that results in loss ofthe reversible terminator and/or premature cleavage of the linker isparticularly problematic, as the unterminated nucleotides will beincorporated during the sequencing reaction, causing some of the growingDNA strands to be extended by two bases rather than one, in what isknown as a dephasing or leading effect.

The modified nucleotides as described herein, e.g., a nucleotide offormulas I, II, or III, were found to be stable (i.e., did not degrade)at 4° C. for at least 7 days. The compounds described herein may conferat least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%,300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 1500%, 2000%, 2500%, or3000% improved stability compared to a nucleotide having a3′-O-azidomethyl protecting group at the same condition for the sameperiod of time. In some embodiments, the stability is measured atambient temperature or a temperature below ambient temperature (e.g.,4-10° C.). In embodiments, the stability is measured at an elevatedtemperature, e.g., 40° C., 45° C., 50° C., 55° C., 60° C., or 65° C. Inembodiments, the stability is measured in solution in a basic pHenvironment, e.g., at pH 9.0, 9.2, 9.4, 9.6, 9.8. or 10.0. In some suchembodiments, the stability is measured with or without the presence ofan enzyme, such as a polymerase (e.g., a DNA polymerase), a terminaldeoxynucleotidyl transferase, or a reverse transcriptase. Inembodiments, the stability is reflected in the out-of-phase metric(e.g., % lead), which directly correlates with the amount of 3′deblocking. In embodiments, the % lead is less than 0.3%. Inembodiments, the % lead is less than 0.2%. In embodiments, the % lead isless than 0.1%.

Example 6. Phasing

As used herein, the term “out-of-phase” refers to phenomena insequencing by synthesis that is caused by incomplete removal of the 3′reversible terminators and fluorophores, and/or failure to completenucleotide incorporation of a portion of DNA strands within clusters fora given sequencing cycle. Sequencing by synthesis of nucleic acidsideally requires the controlled (i.e., one at a time), yet rapid,incorporation of the correct complementary nucleotide opposite theoligonucleotide being sequenced. Following detection, the removal of thereversible terminator leaves a free 3′ hydroxyl group for addition ofthe next nucleotide. If, however, the reversible terminator is removedprematurely, or the solution of reversibly terminated nucleotidescontains impurities (e.g., nucleotides bearing a 3′ hydroxyl group),these unterminated nucleotides may be incorporated into thepolynucleotide. This leads to the clusters of monoclonal amplicons beingout-of-phase, reducing sequencing accuracy and limiting sequencing readlengths.

The unprotected 3′-OH nucleotides could be generated during themanufacturing processes or possibly during the storage and reagenthandling processes. Accordingly, the discovery of nucleotide analogueswhich decrease the incidence of phasing errors provides a greatadvantage in SBS applications over existing nucleotide analogues. Forexample, the nucleotide compounds described herein results in faster SBScycle time, lower out-of-phase values, and permit longer sequencing readlength.

Example 7. Synthesis of Linkers

The process for using polymerase-compatible cleavable moiety containingmolecules generally involves incorporation of a labeled nucleotideanalog into the growing polynucleotide chain, followed by detection ofthe label, then cleavage of the nucleotide analog to remove the covalentmodification blocking continued synthesis (e.g., polymerase-compatiblecleavable moiety). The cleaving step may be accomplished using an enzymeor by chemical cleavage. Modifications of nucleotides may be made on the5′ terminal phosphate or the 3′ hydroxyl group. Developing a trulyreversible set of nucleotide terminators has been a goal for many years.Despite the recent advances only a few solutions have been presented,most of which cause other problems, including inefficient or incompleteincorporation by the polymerase, inefficient or incomplete cleavage ofthe removable group, or harsh conditions needed to for the cleaving stepcausing spurious problems with the remainder of the assay and/orfidelity of the target sequence. Disclosed herein is a new class offluorescently labeled nucleotides that include a new RT bonded to the 3′oxygen.

Experimental Procedures for a Linker:

Sodium iodide (1.5 g, 10.0 mmol) and potassium carbonate (6.9 g, 50mmol) were added to a stirred solution of ethyl 3-hydroxybenzoate (4.15g, 25 mmol), 2-bromomethyl-1,3-dioxolane (10.4 mL, 100 mmol) in DMF (15mL) and was heated to 120° C. The progress of the reaction was monitoredby HPLC (100 mM TEAA/MeCN, 60% to 100% over 15 min, hold for 5 min at100%). The reaction mixture was cooled to room temperature when theamount of ethyl-3-hydroxybenzoate was less than 5%. The suspension wasfiltered and washed with ether (2×50 mL). The combined filtrates werewashed with water (3×50 mL) and brine (50 mL), dried over sodium sulfateand concentrated in vacuo. The crude product was purified by silica gelchromatography (hexanes/ethyl acetate, 80:20) to obtain the desiredcompound, ethyl 3-((1,3-dioxolan-2-yl)methoxy)benzoate as colorlessclear liquid (5.57 g, 88%). ¹H NMR (500 MHz, DMSO) δ 7.59-7.52 (m, 1H),7.48-7.39 (m, 2H), 7.25 (ddd, J=8.3, 2.7, 1.0 Hz, 1H), 5.22 (t, 3.9 Hz,1H), 4.35-4.25 (m, 2H), 4.07 (d, J=3D Hz, 2H), 4.01-3.91 (m, 2H), 3.86(ddd, J=15.2, 9.1, 5.6 Hz, 2H), 1.31 (q, J=12 Hz, 3H); MS: calc'd for[C₁₃H₁₆O₅+Na]: 275.1, found 275.3.

2,4,6-Collidine (2.38 mmol, 3.0 equiv.) was added to a stirred solutionof ethyl 3-((1,3-dioxolan-2-yl)methoxy)benzoate (0.2 g, 0.79 mmol) inDCM (0.1 M) at 0° C. under Ar atmosphere followed by the addition oftrimethylsilyl triflate (1.59 mmol, 2.0 equiv.). The mixture was stirredat the same temperature until the disappearance of an acetal on TLC andformation of highly polar compound was observed, after which potassiumthiotosylate (1.59 mmol, 2.0 equiv.) and 18-crown-6 (1.59 mmol, 2.0equiv.) were added to the reaction mixture. Disappearance of the polarcomponent was confirmed by TLC, after which tert- butyl thiol (1.59mmol, 2.0 equiv.) was added. The reaction mixture was loaded on tosilica gel column upon completion of the reaction and the desiredproduct, ethyl3-(2-(tert-butyldisulfaneyl)-2-(2-hydroxyethoxy)ethoxy)benzoate waseluted with 20% ethyl acetate and hexanes mixture as a colorless oil(235.6 mg, 63% yield). NMR (500 MHz, DMSO) δ 7.57 (dd, J=6.6, 1.2 Hz,1H), 7.51-7.42 (m, 2H), 7.27 (ddd, J=8.2, 2.7, 0.8 Hz, 1H), 4.92 (t,J=5.4 Hz, 1H), 4.69 (t, J=5.4 Hz, 1H), 4.35-4.25 (m, 4H), 3.89-3.80 (m,1H), 3.61-3.49 (m, 3H), 1.37-1.28 (m, 12H). MS: calc'd for[C₁₇H₂₆O₅S₂+Na]: 397.1, found 397.3.

Sodium hydroxide (0.7 mL, 2 M) was added to a stirred solution of ethyl3-(2-(tert-butyldisulfaneyl)-2-(2-hydroxyethoxy)ethoxy)benzoate (131 mg,0.35 mmol) in 1:1 methanol (0.33 mL) and THF (0.33 mL) mixture. Thesolution was initially heterogeneous but became homogenous after 1 hourof stirring. The reaction progress was monitored by HPLC (100 mMTEAA/MeCN, 60% to 100% over 15 min, hold for 5 min at 100%). Uponcompletion, the reaction mixture was concentrated and HCl (1 M, 1.382mL) was added dropwise with stirring until the milky swirl persisted.The aqueous suspension was extracted with DCM (3×15 mL) and the extractswere dried over sodium sulfate. The crude product was purified usingsilica gel chromatography (50% ethyl acetate:hexanes) and3-(2-(tert-butyldisulfaneyl)-2-(2-hydroxyethoxy)ethoxy)benzoic acid wasobtained as a colorless oil (87 mg, 72% yield). ¹H NMR (500 MHz, DMSO) δ13.00 (s, 1H), 7.58-7.52 (m, 1H), 7.47 (dt, J=11.9, 6.1 Hz, 1H), 7.42(t, J=7.9 Hz, 1H), 7.21-7.19 (m, 1H), 4.92 (t, J=5.4 Hz, 1H), 4.69 (s,1H), 4.34-4.24 (m, 2H), 3.88-3.82 (m, 1H), 3.61-3.50 (m, 3H), 1.35-1.26(m, 9H). MS: calc'd for [C₁₅H₂₂O₅S₂+Na]: 369.1, found 369.2.

To a mixture of3-(2-(tert-butyldisulfaneyl)-2-(2-hydroxyethoxy)ethoxy)benzoic acid (43mg, 0.124 mmol), N-(2-aminoethyl)-2,2,2-trifluoroacetamide (28.6 mg,0.148 mmol, 1.2 equiv.), 4-N,N-dimethylaminopyridine (4.5 mg, 0.037mmol, 0.3 equiv.) in DCM (0.2 mL, 0.6 M) at 0° C., was addedA-(3-dimethylaminopropyl)—N′-ethylcarbodiimide hydrochloride (33 mg,0.174 mmol, 1.4 equiv.) in DCM dropwise. The reaction was stirred atroom temperature until the disappearance of the starting material asmonitored by HPLC (100 mM TEAA/MeCN, 60% to 100% over 15 min, hold for 5min at 100%). The reaction mixture was diluted with water and extractedwith ethyl acetate (3×15 mL) and dried over sodium sulfate. The crudewas purified by silica gel chromatography (60% ethyl acetate:hexanes)and3-(2-(A/7-butyldisulfaneyl)-2-(2-hydroxyethoxy)ethoxy)-A-(2-(2,2,2-trifluoroacetamido)ethyl)benzamidewas obtained as colorless liquid (38 mg, 63.3% yield). NMR (500 MHz,DMSO) δ 9.49 (d, J=5.5 Hz, 1H), 8.59 (t, J=5.5 Hz, 1H), 7.40 (dq,J=22.8, 7.7 Hz, 3H), 7.12 (dd, J=8.0, 1.6 Hz, 1H), 4.92 (t, J=5.4 Hz,1H), 4.70 (t, J=5.3 Hz, 1H), 4.35-4.19 (m, 2H), 3.87 (dt, J=9.6, 4.3 Hz,1H), 3.62-3.50 (m, 3H), 3.43-3.33 (m, 4H), 1.31 (s, 9H). MS: calc'd for[C₁₉H₂₇F₃N₂O₅S₂+Na]: 507.1, found 507.2.

To a stirred solution of3-(2-(tert-butyldisulfaneyl)-2-(2-hydroxyethoxy)ethoxy)-N-(2-(2,2,2-trifluoroacetamido)ethyl)benzamide(55 mg, 0.115 mmol) in methanol (0.5 mL), potassium carbonate (45.5 mg,0.329 mmol, 2.9 equiv.) was added. The reaction progress was monitoredby HPLC (100 mM TEAA/MeCN, 60% to 100% over 15 min, hold for 5 min at100%) and upon completion, the reaction mixture was diluted with waterand extracted with ethyl acetate (3×5 mL). The organic fractions werecollected, dried over sodium sulfate and purified by HPLC to obtainA-(2-aminoethyl)-3-(2-(tert-butyldisulfaneyl)-2-(2-hydroxyethoxy)ethoxy)benzamideas colorless liquid. ¹H NMR (500 MHz, DMSO) δ 8.43 (d, J=5.0 Hz, 1H),7.45 (t, J=5.8 Hz, 2H), 7.37 (t, J=7.8 Hz, 1H), 7.11 (dd, 7=7.8, 2.1 Hz,1H), 4.92 (t, 7=5.3 Hz, 1H), 4.71 (s, 1H), 4.28 (d, J=5.9 Hz, 2H), 3.87(dt, J=9.4, 4.2 Hz, 1H), 3.63-3.50 (m, 3H), 3.32-3.22 (m, 4H), 2.71 (t,J=6.5 Hz, 2H), 1.31 (s, 9H). MS: calc'd for [C₁₇H₂₈N₂O₄S₂+H]: 389.2,found 389.4.

2,4,6-Collidine (0.59 mmol, 3.0 equiv.) was added to a stirred solutionof ethyl 3-((1,3-dioxolan-2-yl)methoxy)benzoate (0.05 g, 0.79 mmol) inDCM (0.1 M) at 0° C. under Ar atmosphere followed by the addition oftrimethylsilyl triflate (0.4 mmol, 2.0 equiv.). The mixture was stirredat the same temperature until the disappearance of an acetal on TLC andformation of highly polar compound was observed, after which a solutionof potassium thiocyanate (0.99 mmol, 5.0 equiv.) and 18-crown-6 (0.99mmol, 5.0 equiv.) in acetone (0.2 mL) was added to it. Disappearance ofthe polar component was confirmed by TLC. The product formation wasconfirmed by mass analysis, MS: calc'd for [C₁₄H₁₇NO₅S—H]⁻: 311.0, found310.0.

Example 8. Scarless Sequencing

Typical modified nucleotides were designed based on the rationale thateach of the nucleotides is modified by attaching a unique cleavablefluorophore to the specific location of the base and capping the 3′-OHgroup with a small reversible-terminating moiety so they are stillrecognized by DNA polymerase as substrates. A potential disadvantage ofthis approach is the production of a small molecular “scar” (e.g., apropargylamine or a modified propargylamino moiety) at the nucleotidebase after cleavage of the fluorescent dye from the incorporatednucleotide in the polymerase reaction. The growing DNA chain accumulatesthese scars through each successive round of SBS. In embodimentsdescribed herein, some compounds include a cyclic moiety (e.g.,substituted or unsubstituted cycloalkyl, heterocycloalkyl, aryl, orheteroaryl) off the 3′-oxygen position (i.e., R⁷ of Formula I, II, orIII) permitting the compounds to be “scarless” nucleotide reversibleterminators (NRT) for DNA sequencing by synthesis (SBS). In embodiments,the base of the scarless nucleotides is a monovalent base. The 3′attached reporter indicated by the symbol R⁷, may be fluorescent (e.g.,a coumarin or coumarin derivative; non-limiting examples of coumarinderivatives are provided in Matikonda et al. Chem. Sci., 2020,11,7302-7307, which is incorporated herein by reference). Such novel NRTsmay be employed in a set for use in SBS, wherein each NRT is 3′-Oreversibly blocked and is labeled with a fluorescent dye that has aunique fluorescence emission corresponding to the type of base of eachnucleotide (e.g., a separate emission for A, T, G, and C respectively),thereby installing dual functions (serving as both a reversible blockerand a cleavable fluorescence reporter) to the 3′-O-modified nucleotideanalogues. During SBS, after a nucleotide is incorporated, and thefluorescent reporter imaged, the 3′-O-dye will be cleaved with acleaving agent (cleaving agents may include THPP or TCEP) to generate a3′-OH group that is ready for subsequent extension reactions. Manyfluorescent dye species (several of which are identified herein) aresuitable for polymerase incorporation when attached to the 3′-0 of thesenucleotide analogues.

In embodiments, R⁷ is a coumarin dye or a derivative thereof. Inembodiments, R⁷ is

In embodiments, R⁷ is

wherein z7B is an integer from 0 to 5. In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

wherein R^(7B) is as described herein. In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In embodiments, R⁷ is

In an aspect is provided a compound having the formula:

wherein R¹, B, R⁸, L¹⁰⁰, and R⁴ are as described herein, includingembodiments.

Example 9. Thioaldehyde Stability

The kinetics of a reaction depend on the activation energy, i.e., thedifference between the energy of the reactants and the transition state.However, transition states have only a transitory existence and aredifficult, if not impossible, to observe, isolate, and quantify. Ageneralization to predicting reaction rates is provided in the HammondPostulate, which suggests the activation energy of the rate determiningstep is inversely proportional to the stability of the transition state.In an endothermic reaction the transition state structure is closer tothe structure of the products, and so it follows that a more stableproduct reflects a more stable transition state and has a loweractivation energy.

Invoking the Hammond Postulate for the reaction of interest, i.e., thethiol bearing nucleotide converting to the free 3′OH and a thioaldehyde,depicted in scheme 6, posits the thermodynamic stability of theresultant thioaldehyde influences the cleavage rate. Simplethermodynamics provides the enthalpy changes of the reaction, ΔH, as ameasure of the thermodynamic stability. The enthalpy change iscalculated as the difference in the enthalpy of the products andreactants, ΔH=ΔH_(products)−ΔH_(reactants).

Using ΔH as a corollary for the reaction rate, it is possible to predictwhich reversible terminators will cleave rapidly under suitableconditions. Gas phase calculations were performed using hybrid DensityFunctional Theory (B3LYP) with a large basis set (Valence triple-zetawith two sets of polarization functions); to determine the optimizedstructure and energy of the reactants and the products were performed toderive a ΔH for a variety of compounds, see Table 4. Experimentalevidence supports using ΔH as a proxy for the reaction rate, as reportedin Example 1 and depicted in FIG. 3 , showing the experimentally derivedcleavage halftime for RT #1, RT #2, and RT #3 as a function of thecalculated ΔH. Reducing the energetic burden on the system, i.e.,reducing the enthalpy, corresponds to faster cleavage rates. This isreadily observed in FIG. 3 showing that as the enthalpy decreases for RT#1, RT #2, and RT #3, the cleavage halftime similarly reduces. Inembodiments, the compound has an enthalpy of about 5 to about 12kcal/mol.

TABLE 4 Calculated enthalpies for Scheme 6 reactions. ΔH R⁷ (of Scheme6) Internal Ref No. (kcal/mol)

RT #1 18.0

RT #2 13.5

RT #3 5.6

RT #4 4.1

RT #5 3.6

RT #6 5.4

RT #7 6.6

RT #8 5.8

RT #9 7.9

RT #10 6.8

RT #11 5.7

RT #12 2.5

RT #13 6.3

RT #14 5.5

RT #15 4.9

RT #16 6.3

RT #17 3.8

RT #18 5.3

RT #19 7.2

RT #20 3.7

RT #21 2.2

RT #22 7.2

RT #23 3.0

RT #24 2.7

RT #25 0.7

RT #26 6.7

RT #27 6.3

RT #28 −1.2

RT #29 4.2

RT #30 0.1

RT #31 4.9

RT #32 2.4

RT #33 0.8

RT #34 −1.0

RT #35 2.6

RT #36 7.4

RT #37 4.8

RT #38 5.2

RT #39 3.2

RT #40 7.3

RT #41 5.5

RT #42 4.1

RT #43 3.3

RT #44 6.1

RT #45 8.0

RT #46 5.9

RT #47 2.8

RT #48 0.5

RT #49 6.2

RT #50 9.2

RT #51 6.4

RT #52 6.0

RT #53 10.0

RT #54 10.8

RT #55 9.8

RT #56 11.2

RT #57 −5.6

RT #58 10.6

RT #59 10.2

RT #60 10.5

RT #61 6.6

RT #62 5.6

RT #63 7.9

RT #64 5.8

RT #65 5.1

RT #66 4.9

RT #67 10.3

RT #68 9.3

RT #69 10.0

RT #70 14.7

RT #71 10.4

RT #72 8.1

RT #73 9.2

RT #74 9.3

RT #75 7.1

RT #76 9.1

RT #77 5.3

RT #78 5.4

RT #79 4.6

RT #80 10.6

Numbered Embodiments

The present disclosure provides the following illustrative embodiments.

Embodiment P1. A compound having the formula:

wherein B¹ is a nucleobase;R¹ is independently hydrogen, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂,—CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl, a5′-nucleoside protecting group, monophosphate moiety, polyphosphatemoiety, or nucleic acid moiety; andR² is independently hydrogen, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂,—CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl; or apolymerase-compatible cleavable moiety or an —O-polymerase-compatiblecleavable moiety.

Embodiment P2. The compound of embodiment P1, having the formula:

Embodiment P3. The compound of embodiment P1, having the formula.

Embodiment P4. The compound of any one of embodiments P1 to P3, whereinR² is hydrogen.

Embodiment P5. The compound of any one of embodiments P1 to P4, whereinR′ is —OH, a 5′-nucleoside protecting group, monophosphate moiety,polyphosphate moiety, or nucleic acid moiety.

Embodiment P6. The compound of any one of embodiments P1 to P4, whereinR¹ is a triphosphate moiety.

Embodiment P7. The compound of any one of embodiments P1 to P6, whereinB¹ is a cytosine or a derivative thereof, guanine or a derivativethereof, adenine or a derivative thereof, thymine or a derivativethereof, uracil or a derivative thereof, hypoxanthine or a derivativethereof, xanthine or a derivative thereof, 7-methylguanine or aderivative thereof, 5,6-dihydrouracil or a derivative thereof,5-methylcytosine or a derivative thereof, or 5-hydroxymethylcytosine ora derivative thereof.

Embodiment P8. The compound of any one of embodiments P1 to P6, whereinB¹ is

Embodiment P9. The compound of any one of embodiments P1 to P6, whereinB¹ is

Embodiment P10. The compound of any one of embodiments P1 to P6, whereinB¹ is —B-L¹⁰⁰-R⁴; B is a divalent cytosine or a derivative thereof,divalent guanine or a derivative thereof, divalent adenine or aderivative thereof, divalent thymine or a derivative thereof, divalenturacil or a derivative thereof, divalent hypoxanthine or a derivativethereof, divalent xanthine or a derivative thereof, divalent7-methylguanine or a derivative thereof, divalent 5,6-dihydrouracil or aderivative thereof, divalent 5-methylcytosine or a derivative thereof,or divalent 5-hydroxymethylcytosine or a derivative thereof, L¹⁰⁰ is adivalent linker; and

R⁴ is a detectable moiety.

Embodiment P11. The compound of embodiment P10, wherein B is

Embodiment P12. The compound of embodiment P10 or P11, wherein L¹⁰⁰ is-L¹⁰¹-L¹⁰²-L¹⁰³-L¹⁰⁴-L¹⁰⁵-; L¹⁰¹, L¹⁰², L¹⁰³, L¹⁰⁴, and L¹⁰⁵ areindependently a bond, —NH—, —O—, —C(O)—, —C(O)NH—, —NHC(O)—, —NHC(O)NH—,—C(O)O—, —OC(O)—, substituted or unsubstituted alkylene, substituted orunsubstituted heteroalkylene, substituted or unsubstitutedcycloalkylene, substituted or unsubstituted heterocycloalkylene,substituted or unsubstituted arylene, or substituted or unsubstitutedheteroarylene.

Embodiment P13. The compound of embodiment P10 or P11, wherein L¹⁰⁰ is-L¹⁰¹-O—CH(—SR¹⁰⁰)-L¹⁰³-L¹⁰⁴-L¹⁰⁵-,-L¹⁰¹-O—C(CH₃)(—SR¹⁰⁰)-L¹⁰³-L¹⁰⁴-L¹⁰⁵-, -L¹⁰¹-O—CH(N3)-L¹⁰³-L¹⁰⁴-L¹⁰⁵-,or -L¹⁰¹-O—CH(N₃)—CH₂—O-L¹⁰⁴-L¹⁰⁵-; L¹⁰¹, L¹⁰³, L¹⁰⁴, and L¹⁰⁵ areindependently a bond, —NH—, —O—, —C(O)—, —C(O)NH—, —NHC(O)—, —NHC(O)NH—,—C(O)O—, —OC(O)—, substituted or unsubstituted alkylene, substituted orunsubstituted heteroalkylene, substituted or unsubstitutedcycloalkylene, substituted or unsubstituted heterocycloalkylene,substituted or unsubstituted arylene, or substituted or unsubstitutedheteroarylene; R¹⁰⁰ is —SR¹⁰² or —CN; and R¹⁰² is unsubstituted C₁-C₄alkyl.

Embodiment P14. The compound of embodiment P10 or P11, wherein L¹⁰⁰ is

Embodiment P15. The compound of embodiment P10 or P11, wherein L¹⁰⁰ is

Embodiment P16. The compound of any one of embodiments P1 to P15,wherein R⁴ is

Embodiment P17. The compound of embodiment P10, having the formula:

wherein L¹⁰⁰ is a cleavable linker.

Embodiment P18. A method for sequencing a nucleic acid, comprising: (i)incorporating in series with a nucleic acid polymerase, within areaction vessel, one of four different compounds into a primer to createan extension strand, wherein said primer is hybridized to said nucleicacid and wherein each of the four different compounds comprises a uniquedetectable label; (ii) detecting said unique detectable label of eachincorporated compound, so as to thereby identify each incorporatedcompound in said extension strand, thereby sequencing the nucleic acid;wherein each of said four different compounds is independently acompound of any one of embodiments P1 to P17.

Embodiment P19. A method of incorporating a compound into a primer, themethod comprising combining a polymerase, a primer hybridized to nucleicacid template and the compound within a reaction vessel and allowingsaid polymerase to incorporate said compound into said primer therebyforming an extended primer, wherein said compound is a compound of ofany one of embodiments P1 to P17.

Embodiment P20. A nucleic acid polymerase complex comprising a nucleicacid polymerase, wherein said nucleic acid polymerase is bound to acompound of of any one of embodiments P1 to P17.

Embodiment P21. A compound having the formula:

wherein B¹ is a nucleobase;R¹ is independently hydrogen, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂,—CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl, a5′-O-nucleoside protecting group, monophosphate moiety, polyphosphatemoiety, or nucleic acid moiety;R² is independently hydrogen, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂,—CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl; or apolymerase-compatible cleavable moiety or an —O-polymerase-compatiblecleavable moiety; R⁷ is substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; and R⁸ is unsubstituted C₁-C₆alkyl.

Embodiment P22. The compound of embodiment P21, having the formula:

Embodiment P23. The compound of embodiment P21, having the formula:

Embodiment P24. The compound of any one of embodiments P21 to P23,wherein R² is hydrogen.

Embodiment P25. The compound of any one of embodiments P21 to P24,wherein R¹ is —OH, a 5′-O-nucleoside protecting group, monophosphatemoiety, polyphosphate moiety, or nucleic acid moiety.

Embodiment P26. The compound of any one of embodiments P21 to P24,wherein R¹ is a triphosphate moiety.

Embodiment P27. The compound of any one of embodiments P21 to P26,wherein R⁸ is unsubstituted C₁-C₄ alkyl.

Embodiment P28. The compound of any one of embodiments P21 to P26,wherein R⁸ is unsubstituted methyl or unsubstituted ethyl.

Embodiment P29. The compound of any one of embodiments P21 to P28,wherein B¹ is a cytosine or a derivative thereof, guanine or aderivative thereof, adenine or a derivative thereof, thymine or aderivative thereof, uracil or a derivative thereof, hypoxanthine or aderivative thereof, xanthine or a derivative thereof, 7-methylguanine ora derivative thereof, 5,6-dihydrouracil or a derivative thereof,5-methylcytosine or a derivative thereof, or 5-hydroxymethylcytosine ora derivative thereof.

Embodiment P30. The compound of any one of embodiments P21 to P28,wherein B¹ is

Embodiment P31. The compound of any one of embodiments P21 to P28,wherein B¹ is

Embodiment P32. The compound of any one of embodiments P21 to P28,wherein B¹ is —B-L¹⁰⁰-R⁴; B is a divalent cytosine or a derivativethereof, divalent guanine or a derivative thereof, divalent adenine or aderivative thereof, divalent thymine or a derivative thereof, divalenturacil or a derivative thereof, divalent hypoxanthine or a derivativethereof, divalent xanthine or a derivative thereof, divalent7-methylguanine or a derivative thereof, divalent 5,6-dihydrouracil or aderivative thereof, divalent 5-methylcytosine or a derivative thereof,or divalent 5-hydroxymethylcytosine or a derivative thereof, L¹⁰⁰ is adivalent linker; and R⁴ is a detectable moiety.

Embodiment P33. The compound of embodiment P32, wherein B is

Embodiment P34. The compound of embodiment P32 or P33, wherein L¹⁰⁰ is-L¹⁰¹-L¹⁰²-L¹⁰³-L¹⁰⁴-L¹⁰⁵-; L¹⁰¹, L¹⁰², L¹⁰³, L¹⁰⁴, and L¹⁰⁵ areindependently a bond, —NH—, —O—, —C(O)—, —C(O)NH—, —NHC(O)—, —NHC(O)NH—,—C(O)O—, —OC(O)—, substituted or unsubstituted alkylene, substituted orunsubstituted heteroalkylene, substituted or unsubstitutedcycloalkylene, substituted or unsubstituted heterocycloalkylene,substituted or unsubstituted arylene, or substituted or unsubstitutedheteroarylene.

Embodiment P35. The compound of embodiment P32 or P33, wherein L¹⁰⁰ is-L¹⁰¹-O—CH(—SR¹⁰⁰)-L¹⁰³-L¹⁰⁴-L¹⁰⁵-,-L¹⁰¹-O—C(CH₃)(—SR¹⁰⁰)-L¹⁰³-L¹⁰⁴-L¹⁰⁵-, -L¹⁰¹-O—CH(N)L¹⁰³-L¹⁰⁴-L¹⁰⁵-, or-L¹⁰¹-O—CH(N₃)—CH₂—O-L¹⁰⁴-L¹⁰⁵-; L¹⁰¹, L¹⁰³, L¹⁰⁴, and L¹⁰⁵ areindependently a bond, —NH—, —O—, —C(O)—, —C(O)NH—, —NHC(O)—, —NHC(O)NH—,—C(O)O—, —OC(O)—, substituted or unsubstituted alkylene, substituted orunsubstituted heteroalkylene, substituted or unsubstitutedcycloalkylene, substituted or unsubstituted heterocycloalkylene,substituted or unsubstituted arylene, or substituted or unsubstitutedheteroarylene; R¹⁰⁰ is —SR¹⁰² or —CN; and R¹⁰² is unsubstituted C₁-C₄alkyl.

Embodiment P36. The compound of embodiment P32 or P33, wherein L¹⁰⁰ is

Embodiment P37. The compound of embodiment P32 or P33, wherein L¹⁰⁰ is

Embodiment P38. The compound of any one of embodiments P21 to P37,wherein R⁷ is substituted or unsubstituted aryl.

Embodiment P39. The compound of any one of embodiments P21 to P37,wherein R⁷ is unsubstituted aryl.

Embodiment P40. The compound of any one of embodiments P21 to P37,wherein R⁷ is unsubstituted phenyl.

Embodiment P41. The compound of any one of embodiments P21 to P37,wherein R⁷ is substituted or unsubstituted heteroaryl.

Embodiment P42. The compound of any one of embodiments P21 to P37,wherein R⁷ is substituted or unsubstituted 5 to 6 membered heteroaryl.

Embodiment P43. The compound of embodiment P32, having the formula:

wherein L¹⁰⁰ is a cleavable linker.

Embodiment P44. The compound of embodiment P43, wherein R⁷ issubstituted or unsubstituted aryl.

Embodiment P45. The compound of embodiment P43, wherein R⁷ issubstituted or unsubstituted heteroaryl.

Embodiment P46. The compound of embodiment P32, having the formula:

wherein L¹⁰⁰ is a cleavable linker.

Embodiment P47. The compound of embodiment P32, having the formula:

wherein L¹⁰⁰ is a cleavable linker.

Embodiment P48. A method for sequencing a nucleic acid, comprising: (i)incorporating in series with a nucleic acid polymerase, within areaction vessel, one of four different compounds into a primer to createan extension strand, wherein said primer is hybridized to said nucleicacid and wherein each of the four different compounds comprises a uniquedetectable label; (ii) detecting said unique detectable label of eachincorporated compound, so as to thereby identify each incorporatedcompound in said extension strand, thereby sequencing the nucleic acid;wherein each of said four different compounds is independently acompound of any one of embodiments P21 to P47.

Embodiment P49. A method of incorporating a compound into a primer, themethod comprising combining a polymerase, a primer hybridized to nucleicacid template and the compound within a reaction vessel and allowingsaid polymerase to incorporate said compound into said primer therebyforming an extended primer, wherein said compound is a compound of anyone of embodiments P21 to P47.

Embodiment P50. A nucleic acid polymerase complex comprising a nucleicacid polymerase, wherein said nucleic acid polymerase is bound to acompound of any one of embodiments P21 to P47.

Additional Embodiments

Embodiment 1. A compound having the formula:

wherein B¹ is a nucleobase; R¹ is independently a polyphosphate moiety,5′-O-nucleoside protecting group, monophosphate moiety, nucleic acidmoiety, hydrogen, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂, —CHBr₂,—CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂, —COOH,—CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂,—NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃, —OCF₃, —OCBr₃,—OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl, —OCH₂Br, —OCH₂I,—OCH₂F, —N₃, —SF₅, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl; R² isindependently hydrogen, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂,—CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl; or apolymerase-compatible cleavable moiety; R⁷ is unsubstituted orsubstituted aryl, or substituted or unsubstituted heteroaryl; and R⁸ isunsubstituted C₁-C₆ alkyl.

Embodiment 2. The compound of Embodiment 1, having the formula:

Embodiment 3. The compound of Embodiment 1, having the formula:

Embodiment 4. The compound of any one of Embodiments 1 to 3, wherein R²is hydrogen.

Embodiment 5. The compound of any one of Embodiments 1 to 4, wherein R¹is —OH, a 5′-O-nucleoside protecting group, monophosphate moiety,polyphosphate moiety, or nucleic acid moiety.

Embodiment 6. The compound of any one of Embodiments 1 to 4, wherein R¹is a triphosphate moiety.

Embodiment 7. The compound of any one of Embodiments 1 to 6, wherein R⁸is unsubstituted C₁-C₄ alkyl.

Embodiment 8. The compound of any one of Embodiments 1 to 6, wherein R⁸is unsubstituted methyl or unsubstituted ethyl.

Embodiment 9. The compound of any one of Embodiments 1 to 8, wherein B¹is a cytosine or a derivative thereof, guanine or a derivative thereof,adenine or a derivative thereof, thymine or a derivative thereof, uracilor a derivative thereof, hypoxanthine or a derivative thereof, xanthineor a derivative thereof, 7-methylguanine or a derivative thereof,5,6-dihydrouracil or a derivative thereof, 5-methylcytosine or aderivative thereof, or 5-hydroxymethylcytosine or a derivative thereof.

Embodiment 10. The compound of any one of Embodiments 1 to 8, wherein B¹is

Embodiment 11. The compound of any one of Embodiments 1 to 8, wherein B¹is

Embodiment 12. The compound of any one of Embodiments 1 to 8, wherein B¹is —B-L¹⁰⁰-R⁴; B is a divalent cytosine or a derivative thereof,divalent guanine or a derivative thereof, divalent adenine or aderivative thereof, divalent thymine or a derivative thereof, divalenturacil or a derivative thereof, divalent hypoxanthine or a derivativethereof, divalent xanthine or a derivative thereof, divalent7-methylguanine or a derivative thereof, divalent 5,6-dihydrouracil or aderivative thereof, divalent 5-methylcytosine or a derivative thereof,or divalent 5-hydroxymethylcytosine or a derivative thereof, L¹⁰⁰ is adivalent linker; and R⁴ is a detectable moiety.

Embodiment 13. The compound of Embodiment 12, wherein B is

Embodiment 14. The compound of Embodiment 12 or 13, wherein L¹⁰⁰ is adivalent linker comprising

wherein, R⁹ is independently substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl.

Embodiment 15. The compound of Embodiment 12 or 13, wherein L¹⁰⁰ is adivalent linker comprising

wherein, R¹⁰² is unsubstituted C₁-C₄ alkyl.

Embodiment 16. The compound of Embodiments 12 or 13, wherein L¹⁰⁰ is-L¹⁰¹-L¹⁰²-L¹⁰³-L¹⁰⁴-L¹⁰⁵-; L¹⁰¹, L¹⁰², L¹⁰³, L¹⁰⁴, and L¹⁰⁵ areindependently a bond, —NH—, —O—, —C(O)—, —C(O)NH—, —NHC(O)—, —NHC(O)NH—,—C(O)O—, —OC(O)—, substituted or unsubstituted alkylene, substituted orunsubstituted heteroalkylene, substituted or unsubstitutedcycloalkylene, substituted or unsubstituted heterocycloalkylene,substituted or unsubstituted arylene, or substituted or unsubstitutedheteroarylene.

Embodiment 17. The compound of Embodiments 12 or 13, wherein L¹⁰⁰ is-L¹⁰¹-O—CH(—SR¹⁰⁰)-L¹⁰³-L¹⁰⁴-L¹⁰⁵-,-L¹⁰¹-O—C(CH₃)(—SR¹⁰⁰)-L¹⁰³-L¹⁰⁴-L¹⁰⁵-, -L¹⁰¹-O—CH(N₃)-L⁰³-L¹⁰⁴-L¹⁰⁵-,or -L¹⁰¹-O—CH(N₃)—CH₂—O-L¹⁰⁴-L¹⁰⁵-; L¹⁰¹, L¹⁰³, L¹⁰⁴, and L¹⁰⁵ areindependently a bond, —NH—, —O—, —C(O)—, —C(O)NH—, —NHC(O)—, —NHC(O)NH—,—C(O)O—, —OC(O)—, substituted or unsubstituted alkylene, substituted orunsubstituted heteroalkylene, substituted or unsubstitutedcycloalkylene, substituted or unsubstituted heterocycloalkylene,substituted or unsubstituted arylene, or substituted or unsubstitutedheteroarylene; R¹⁰⁰ is —SR¹⁰² or —CN; and R¹⁰² is unsubstituted C₁-C₄alkyl.

Embodiment 18. The compound of Embodiment 16, wherein L¹⁰⁰ is

Embodiment 19. The compound of Embodiment 16, wherein L¹⁰⁰ is

Embodiment 20. The compound of Embodiment 12 or 13, wherein L¹⁰⁰ is

Embodiment 21. The compound of Embodiment 12 or 13, wherein L¹⁰⁰ is

Embodiment 22. The compound of any one of Embodiments 1 to 21, whereinR⁷ is substituted or unsubstituted aryl.

Embodiment 23. The compound of any one of Embodiments 1 to 21, whereinR⁷ is unsubstituted aryl.

Embodiment 24. The compound of any one of Embodiments 1 to 21, whereinR⁷ is unsubstituted phenyl.

Embodiment 25. The compound of any one of Embodiments 1 to 21, whereinR⁷ is substituted or unsubstituted heteroaryl.

Embodiment 26. The compound of any one of Embodiments 1 to 21, whereinR⁷ is substituted or unsubstituted 5 to 6 membered heteroaryl.

Embodiment 27. The compound of any one of Embodiments 1 to 21, whereinR⁷ is substituted or unsubstituted 5 to 6 membered heteroaryl.

Embodiment 28. The compound of any one of Embodiments 1 to 21, whereinR⁷ is

Embodiment 29. The compound of Embodiment 12, having the formula:

wherein L¹⁰⁰ is a cleavable linker.

Embodiment 30. The compound of Embodiment 29, wherein R⁷ is substitutedor unsubstituted aryl.

Embodiment 31. The compound of Embodiment 29, wherein R⁷ is substitutedor unsubstituted heteroaryl.

Embodiment 32. The compound of Embodiment 29, wherein R⁷ is

Embodiment 33. The compound of Embodiment 12, having the formula:

wherein L¹⁰⁰ is a cleavable linker.

Embodiment 34. The compound of Embodiment 33, wherein L¹⁰⁰ is

Embodiment 35. The compound of Embodiment 33, wherein L¹⁰⁰ is

Embodiment 36. The compound of Embodiment 33, wherein L¹⁰⁰ is

Embodiment 37. The compound of Embodiment 12, having the formula.

wherein L¹⁰⁰ is a cleavable linker.

Embodiment 38. The compound of Embodiment 37, wherein L¹⁰⁰ is

Embodiment 39. The compound of Embodiment 37, wherein L¹⁰⁰ is

Embodiment 40. The compound of Embodiment 37, wherein L¹⁰⁰ is

Embodiment 41. A method for sequencing a nucleic acid, comprising: i)incorporating in series with a nucleic acid polymerase, within areaction vessel, one of four different compounds into a primer to createan extension strand, wherein said primer is hybridized to said nucleicacid and wherein each of the four different compounds comprises a uniquedetectable label; ii) detecting said unique detectable label of eachincorporated compound, so as to thereby identify each incorporatedcompound in said extension strand, thereby sequencing the nucleic acid;wherein each of said four different compounds is independently acompound of any one of Embodiments 1 to 37.

Embodiment 42. A method of incorporating a compound into a primer, themethod comprising combining a polymerase, a primer hybridized to nucleicacid template and the compound within a reaction vessel and allowingsaid polymerase to incorporate said compound into said primer therebyforming an extended primer, wherein said compound is a compound of anyone of Embodiments 1 to 37.

Embodiment 43. A nucleic acid polymerase complex comprising a nucleicacid polymerase, wherein said nucleic acid polymerase is bound to acompound of any one of Embodiments 1 to 37.

1. A compound having the formula:

wherein B¹ is a nucleobase; R¹ is a polyphosphate moiety,5′-O-nucleoside protecting group, monophosphate moiety, nucleic acidmoiety, hydrogen, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂, —CHBr₂,—CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂, —COOH,—CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂,—NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃, —OCF₃, —OCBr₃,—OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl, —OCH₂Br, —OCH₂I,—OCH₂F, —N₃, —SF₅, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl; R² ishydrogen, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂, —CHBr₂, —CHF₂,—CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂, —COOH, —CONH₂,—NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂,—NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃, —OCF₃, —OCBr₃,—OCI₃, -OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl, —OCH₂Br, —OCH₂I,—OCH₂F, —N₃, —SF₅, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl; or apolymerase-compatible cleavable moiety; R⁷ is unsubstituted orsubstituted cycloalkyl, or substituted or unsubstitutedheterocycloalkyl; and R⁸ is unsubstituted C₁-C₆ alkyl.
 2. The compoundof claim 1, wherein R² is hydrogen.
 3. The compound of claim 1, whereinR¹ is —OH, a 5′-O-nucleoside protecting group, monophosphate moiety,polyphosphate moiety, or nucleic acid moiety.
 4. The compound of claim1, wherein R¹ is a triphosphate moiety.
 5. The compound of claim 1,wherein R⁸ is unsubstituted C₁-C₄ alkyl.
 6. The compound of claim 1,wherein R⁸ is unsubstituted methyl or unsubstituted ethyl.
 7. Thecompound of claim 1, wherein B¹ is a cytosine or a derivative thereof,guanine or a derivative thereof, adenine or a derivative thereof,thymine or a derivative thereof, uracil or a derivative thereof,hypoxanthine or a derivative thereof, xanthine or a derivative thereof,7-methylguanine or a derivative thereof, 5,6-dihydrouracil or aderivative thereof, 5-methylcytosine or a derivative thereof, or5-hydroxymethylcytosine or a derivative thereof.
 8. The compound ofclaim 1, wherein B¹ is


9. The compound of claim 1, wherein B¹ is —B-L¹⁰⁰-R⁴; B is a divalentcytosine or a derivative thereof, divalent guanine or a derivativethereof, divalent adenine or a derivative thereof, divalent thymine or aderivative thereof, divalent uracil or a derivative thereof, divalenthypoxanthine or a derivative thereof, divalent xanthine or a derivativethereof, divalent 7-methylguanine or a derivative thereof, divalent5,6-dihydrouracil or a derivative thereof, divalent 5-methylcytosine ora derivative thereof, or divalent 5-hydroxymethylcytosine or aderivative thereof, L¹⁰⁰ is a divalent linker; and R⁴ is a detectablemoiety.
 10. The compound of claim 9, wherein L¹⁰⁰ is a divalent linkercomprising

wherein R⁹ is substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl.
 11. Thecompound of claim 9, wherein L¹⁰⁰ is a divalent linker comprising

wherein R¹⁰² is unsubstituted C₁-C₄ alkyl.
 12. The compound of claim 9,wherein L¹⁰⁰ is -L¹⁰¹-L¹⁰²-L¹⁰³-L¹⁰⁴-L¹⁰⁵-; L¹⁰¹, L¹⁰², L¹⁰³, L¹⁰⁴, andL¹⁰⁵ are independently a bond, —NH—, —O—, —C(O)—, —C(O)NH—, —NHC(O)—,—NHC(O)NH—, —C(O)O—, —OC(O)—, substituted or unsubstituted alkylene,substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene.
 13. The compound of claim12, wherein L¹⁰⁰ is

wherein R⁹ is substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl; and R¹⁰²is unsubstituted C₁-C₄ alkyl.
 14. The compound of claim 12, wherein L¹⁰⁰is

wherein R⁹ is substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl; and R¹⁰²is unsubstituted C₁-C₄ alkyl.
 15. The compound of claim 9, wherein L¹⁰⁰is


16. The compound of claim 18, wherein L¹⁰⁰ is


17. The compound of claim 1, wherein R⁷ is substituted or unsubstitutedcycloalkyl.
 18. The compound of claim 1, wherein R⁷ is unsubstitutedcycloalkyl.
 19. The compound of claim 1, wherein R⁷ is substituted orunsubstituted heterocycloalkyl.
 20. (canceled)
 21. The compound of claim9, having the formula:

wherein L¹⁰⁰ is a cleavable linker.
 22. The compound of claim 21,wherein R⁷ is substituted or unsubstituted cycloalkyl.
 23. The compoundof claim 21, wherein R⁷ is substituted or unsubstitutedheterocycloalkyl.
 24. (canceled)
 25. The compound of claim 21, whereinL¹⁰⁰ is

wherein R⁹ is substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl; R¹⁰² isunsubstituted C₁-C₄ alkyl; and L¹⁰³, L¹⁰⁴, and L¹⁰⁵ are independently abond, —NH—, —O—, —C(O)—, —C(O)NH—, —NHC(O)—, —NHC(O)NH—, —C(O)O—,—OC(O)—, substituted or unsubstituted alkylene, substituted orunsubstituted heteroalkylene, substituted or unsubstitutedcycloalkylene, substituted or unsubstituted heterocycloalkylene,substituted or unsubstituted arylene, or substituted or unsubstitutedheteroarylene.
 26. (canceled)
 27. (canceled)
 28. A method for sequencinga nucleic acid, comprising: (i) incorporating in series with a nucleicacid polymerase, within a reaction vessel, one of four differentcompounds into a primer to create an extension strand, wherein saidprimer is hybridized to said nucleic acid and wherein each of the fourdifferent compounds comprises a unique detectable label; (ii) detectingsaid unique detectable label of each incorporated compound, so as tothereby identify each incorporated compound in said extension strand,thereby sequencing the nucleic acid; wherein each of said four differentcompounds is independently a compound of claim
 1. 29. A method ofincorporating a compound into a primer, the method comprising combininga polymerase, a primer hybridized to nucleic acid template and thecompound within a reaction vessel and allowing said polymerase toincorporate said compound into said primer thereby forming an extendedprimer, wherein said compound is a compound of claim
 1. 30. A nucleicacid polymerase complex comprising a nucleic acid polymerase, whereinsaid nucleic acid polymerase is bound to a compound of claim
 1. 31. Thecompound of claim 1, wherein R⁷ is unsubstituted cycloalkyl orunsubstituted heterocycloalkyl.
 32. The compound of claim 1, wherein R⁷is